<?xml version="1.0" encoding="us-ascii"?><InstanceReport xmlns:xsd="http://www.w3.org/2001/XMLSchema" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance"><Version>2.4.0.8</Version><ReportLongName>100700 - Disclosure - Investment in Affiliate</ReportLongName><DisplayLabelColumn>true</DisplayLabelColumn><ShowElementNames>false</ShowElementNames><RoundingOption /><HasEmbeddedReports>false</HasEmbeddedReports><Columns><Column FlagID="0"><Id>1</Id><IsAbstractGroupTitle>false</IsAbstractGroupTitle><LabelSeparator>

</LabelSeparator><CurrencyCode /><FootnoteIndexer /><hasSegments>false</hasSegments><hasScenarios>false</hasScenarios><MCU><KeyName /><CurrencySymbol /><contextRef><ContextID>Q2_2013_Dur</ContextID><EntitySchema>http://www.sec.gov/CIK</EntitySchema><EntityValue>0001401112</EntityValue><PeriodDisplayName /><PeriodType>duration</PeriodType><PeriodStartDate>2013-01-01T00:00:00</PeriodStartDate><PeriodEndDate>2013-06-30T00:00:00</PeriodEndDate><Segments /><Scenarios /></contextRef><UPS /><CurrencyCode /><OriginalCurrencyCode /></MCU><CurrencySymbol /><Labels><Label Key="CalendarSupplement" Id="0" Label="6 Months Ended" /><Label Key="Calendar" Id="1" Label="Jun. 30, 2013" /></Labels></Column></Columns><Rows><Row FlagID="0"><Id>1</Id><IsAbstractGroupTitle>true</IsAbstractGroupTitle><LabelSeparator>

</LabelSeparator><Level>1</Level><ElementName>us-gaap_InvestmentsInAffiliatesSubsidiariesAssociatesAndJointVenturesAbstract</ElementName><ElementPrefix>us-gaap_</ElementPrefix><IsBaseElement>true</IsBaseElement><BalanceType>na</BalanceType><PeriodType>duration</PeriodType><IsReportTitle>false</IsReportTitle><IsSegmentTitle>false</IsSegmentTitle><IsCalendarTitle>false</IsCalendarTitle><IsEquityPrevioslyReportedAsRow>false</IsEquityPrevioslyReportedAsRow><IsEquityAdjustmentRow>false</IsEquityAdjustmentRow><IsBeginningBalance>false</IsBeginningBalance><IsEndingBalance>false</IsEndingBalance><IsReverseSign>false</IsReverseSign><FootnoteIndexer /><Cells><Cell FlagID="0" ContextID="" UnitID=""><Id>1</Id><IsNumeric>false</IsNumeric><IsRatio>false</IsRatio><DisplayZeroAsNone>false</DisplayZeroAsNone><NumericAmount>0</NumericAmount><RoundedNumericAmount>0</RoundedNumericAmount><NonNumbericText /><FootnoteIndexer /><CurrencyCode /><CurrencySymbol /><IsIndependantCurrency>false</IsIndependantCurrency><ShowCurrencySymbol>false</ShowCurrencySymbol><DisplayDateInUSFormat>false</DisplayDateInUSFormat></Cell></Cells><ElementDataType>xbrli:stringItemType</ElementDataType><SimpleDataType>string</SimpleDataType><IsTotalLabel>false</IsTotalLabel><UnitID>0</UnitID><Label>Investment in Affiliate [Abstract]</Label></Row><Row FlagID="0"><Id>2</Id><IsAbstractGroupTitle>false</IsAbstractGroupTitle><LabelSeparator>

</LabelSeparator><Level>2</Level><ElementName>us-gaap_InvestmentsInAndAdvancesToAffiliatesTextBlock</ElementName><ElementPrefix>us-gaap_</ElementPrefix><IsBaseElement>true</IsBaseElement><BalanceType>na</BalanceType><PeriodType>duration</PeriodType><IsReportTitle>false</IsReportTitle><IsSegmentTitle>false</IsSegmentTitle><IsCalendarTitle>false</IsCalendarTitle><IsEquityPrevioslyReportedAsRow>false</IsEquityPrevioslyReportedAsRow><IsEquityAdjustmentRow>false</IsEquityAdjustmentRow><IsBeginningBalance>false</IsBeginningBalance><IsEndingBalance>false</IsEndingBalance><IsReverseSign>false</IsReverseSign><PreferredLabelRole>terseLabel</PreferredLabelRole><FootnoteIndexer /><Cells><Cell FlagID="0" ContextID="Q2_2013_Dur" UnitID=""><Id>1</Id><IsNumeric>false</IsNumeric><IsRatio>false</IsRatio><DisplayZeroAsNone>false</DisplayZeroAsNone><NumericAmount>0</NumericAmount><RoundedNumericAmount>0</RoundedNumericAmount><NonNumbericText>&lt;div&gt;&lt;p style="margin-left:0pt;margin-right:0pt;margin-top:0pt;margin-bottom:.0001pt;font-size:10.0pt;font-family:&amp;quot;Times New Roman&amp;quot;;"&gt;&lt;b&gt;7. Investment in Affiliate&lt;!--Exclude empty div--&gt;&lt;/b&gt;&lt;!--Exclude empty p--&gt;&lt;/p&gt;&lt;p style="margin-left:0pt;margin-right:0pt;margin-top:0pt;margin-bottom:.0001pt;font-size:10.0pt;font-family:&amp;quot;Times New Roman&amp;quot;;"&gt;&amp;#160;&lt;!--Exclude empty p--&gt;&lt;/p&gt;&lt;p style="margin-left:0pt;margin-right:0pt;margin-top:0pt;margin-bottom:.0001pt;font-size:10.0pt;font-family:&amp;quot;Times New Roman&amp;quot;;text-align:justify;text-justify:inter-ideograph;"&gt;The following table is a reconciliation of the Company&amp;#8217;s investment in affiliate as presented on the accompanying unaudited interim condensed consolidated balance sheet:&lt;!--Exclude empty div--&gt;&lt;!--Exclude empty div--&gt;&lt;!--Exclude empty p--&gt;&lt;/p&gt;&lt;p style="margin-left:0pt;margin-right:0pt;margin-top:0pt;margin-bottom:.0001pt;font-size:10.0pt;font-family:&amp;quot;Times New Roman&amp;quot;;text-align:justify;text-justify:inter-ideograph;"&gt;&lt;!--Exclude empty div--&gt;&amp;#160;&lt;!--Exclude empty p--&gt;&lt;/p&gt;&lt;div align="center"&gt;&lt;table cellspacing="0" style="vertical-align:top;font-family:Georgia;;border-collapse:collapse;margin-left:0pt;"&gt;&lt;tr style=";height:14.15pt;"&gt;&lt;td style="vertical-align:top;font-family:Georgia;;width:329.4pt;vertical-align:middle;border-top:solid windowtext 0.5pt;border-bottom:solid windowtext 0.5pt;border-top:solid windowtext 0.5pt;border-bottom:solid windowtext 0.5pt;padding:0pt 5.4pt 0pt 5.4pt;"&gt;&lt;p style="margin-left:0pt;margin-right:0pt;margin-top:0pt;margin-bottom:.0001pt;font-size:10.0pt;font-family:&amp;quot;Times New Roman&amp;quot;;"&gt;&lt;b&gt;Balance, December 31, 2012&lt;!--Exclude empty div--&gt;&lt;/b&gt;&lt;!--Exclude empty p--&gt;&lt;/p&gt;&lt;/td&gt;&lt;td style="vertical-align:top;font-family:Georgia;;width:95.4pt;vertical-align:middle;border-top:solid windowtext 0.5pt;border-bottom:solid windowtext 0.5pt;border-top:solid windowtext 0.5pt;border-bottom:solid windowtext 0.5pt;padding:0pt 5.4pt 0pt 5.4pt;"&gt;&lt;p style="margin-left:0pt;margin-right:0pt;margin-top:0pt;margin-bottom:.0001pt;font-size:10.0pt;font-family:&amp;quot;Times New Roman&amp;quot;;text-align:right;"&gt;&lt;b&gt;$19,987,743&lt;!--Exclude empty div--&gt;&lt;/b&gt;&lt;!--Exclude empty div--&gt;&lt;!--Exclude empty p--&gt;&lt;/p&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr style=";height:14.15pt;"&gt;&lt;td style="vertical-align:top;font-family:Georgia;;width:329.4pt;vertical-align:middle;border-top:solid windowtext 0.5pt;border-top:solid windowtext 0.5pt;padding:0pt 5.4pt 0pt 5.4pt;"&gt;&lt;p style="margin-left:0pt;margin-right:0pt;margin-top:0pt;margin-bottom:.0001pt;font-size:10.0pt;font-family:&amp;quot;Times New Roman&amp;quot;;"&gt;Equity in net income of affiliate&lt;!--Exclude empty div--&gt;&lt;!--Exclude empty p--&gt;&lt;/p&gt;&lt;/td&gt;&lt;td style="vertical-align:top;font-family:Georgia;;width:95.4pt;vertical-align:middle;border-top:solid windowtext 0.5pt;border-top:solid windowtext 0.5pt;padding:0pt 5.4pt 0pt 5.4pt;"&gt;&lt;p style="margin-left:0pt;margin-right:0pt;margin-top:0pt;margin-bottom:.0001pt;font-size:10.0pt;font-family:&amp;quot;Times New Roman&amp;quot;;text-align:right;"&gt;978,702&lt;!--Exclude empty div--&gt;&lt;!--Exclude empty div--&gt;&lt;!--Exclude empty p--&gt;&lt;/p&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr style=";height:14.15pt;"&gt;&lt;td style="vertical-align:top;font-family:Georgia;;width:329.4pt;vertical-align:middle;padding:0pt 5.4pt 0pt 5.4pt;"&gt;&lt;p style="margin-left:0pt;margin-right:0pt;margin-top:0pt;margin-bottom:.0001pt;font-size:10.0pt;font-family:&amp;quot;Times New Roman&amp;quot;;"&gt;Equity in other comprehensive income of affiliate&lt;!--Exclude empty div--&gt;&lt;!--Exclude empty p--&gt;&lt;/p&gt;&lt;/td&gt;&lt;td style="vertical-align:top;font-family:Georgia;;width:95.4pt;vertical-align:middle;padding:0pt 5.4pt 0pt 5.4pt;"&gt;&lt;p style="margin-left:0pt;margin-right:0pt;margin-top:0pt;margin-bottom:.0001pt;font-size:10.0pt;font-family:&amp;quot;Times New Roman&amp;quot;;text-align:right;"&gt;106,709&lt;!--Exclude empty div--&gt;&lt;!--Exclude empty div--&gt;&lt;!--Exclude empty p--&gt;&lt;/p&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr style=";height:14.15pt;"&gt;&lt;td style="vertical-align:top;font-family:Georgia;;width:329.4pt;vertical-align:middle;padding:0pt 5.4pt 0pt 5.4pt;"&gt;&lt;p style="margin-left:0pt;margin-right:0pt;margin-top:0pt;margin-bottom:.0001pt;font-size:10.0pt;font-family:&amp;quot;Times New Roman&amp;quot;;"&gt;Dividends received&lt;!--Exclude empty div--&gt;&lt;!--Exclude empty p--&gt;&lt;/p&gt;&lt;/td&gt;&lt;td style="vertical-align:top;font-family:Georgia;;width:95.4pt;vertical-align:middle;padding:0pt 5.4pt 0pt 5.4pt;"&gt;&lt;p style="margin-left:0pt;margin-right:0pt;margin-top:0pt;margin-bottom:.0001pt;font-size:10.0pt;font-family:&amp;quot;Times New Roman&amp;quot;;text-align:right;"&gt;(1,168,750)&lt;!--Exclude empty div--&gt;&lt;!--Exclude empty div--&gt;&lt;!--Exclude empty p--&gt;&lt;/p&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr style=";height:14.15pt;"&gt;&lt;td style="vertical-align:top;font-family:Georgia;;width:329.4pt;vertical-align:middle;padding:0pt 5.4pt 0pt 5.4pt;"&gt;&lt;p style="margin-left:0pt;margin-right:0pt;margin-top:0pt;margin-bottom:.0001pt;font-size:10.0pt;font-family:&amp;quot;Times New Roman&amp;quot;;"&gt;Loss on investment in affiliate&lt;!--Exclude empty div--&gt;&lt;!--Exclude empty p--&gt;&lt;/p&gt;&lt;/td&gt;&lt;td style="vertical-align:top;font-family:Georgia;;width:95.4pt;vertical-align:middle;padding:0pt 5.4pt 0pt 5.4pt;"&gt;&lt;p style="margin-left:0pt;margin-right:0pt;margin-top:0pt;margin-bottom:.0001pt;font-size:10.0pt;font-family:&amp;quot;Times New Roman&amp;quot;;text-align:right;"&gt;(390,821)&lt;!--Exclude empty div--&gt;&lt;!--Exclude empty div--&gt;&lt;!--Exclude empty p--&gt;&lt;/p&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr style=";height:14.15pt;"&gt;&lt;td style="vertical-align:top;font-family:Georgia;;width:329.4pt;vertical-align:middle;border-top:solid windowtext 0.5pt;border-bottom:solid windowtext 0.5pt;border-top:solid windowtext 0.5pt;border-bottom:solid windowtext 0.5pt;padding:0pt 5.4pt 0pt 5.4pt;"&gt;&lt;p style="margin-left:0pt;margin-right:0pt;margin-top:0pt;margin-bottom:.0001pt;font-size:10.0pt;font-family:&amp;quot;Times New Roman&amp;quot;;"&gt;&lt;b&gt;Balance, June 30, 2013&lt;!--Exclude empty div--&gt;&lt;/b&gt;&lt;!--Exclude empty p--&gt;&lt;/p&gt;&lt;/td&gt;&lt;td style="vertical-align:top;font-family:Georgia;;width:95.4pt;vertical-align:middle;border-top:solid windowtext 0.5pt;border-bottom:solid windowtext 0.5pt;border-top:solid windowtext 0.5pt;border-bottom:solid windowtext 0.5pt;padding:0pt 5.4pt 0pt 5.4pt;"&gt;&lt;p style="margin-left:0pt;margin-right:0pt;margin-top:0pt;margin-bottom:.0001pt;font-size:10.0pt;font-family:&amp;quot;Times New Roman&amp;quot;;text-align:right;"&gt;&lt;b&gt;$19,513,583&lt;!--Exclude empty div--&gt;&lt;/b&gt;&lt;!--Exclude empty div--&gt;&lt;!--Exclude empty p--&gt;&lt;/p&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td style="margin:0;padding:0;border:none;width:329.4pt;" /&gt;&lt;td style="margin:0;padding:0;border:none;width:95.4pt;" /&gt;&lt;/tr&gt;&lt;/table&gt;&lt;/div&gt;&lt;p style="margin-left:0pt;margin-right:0pt;margin-top:0pt;margin-bottom:.0001pt;font-size:10.0pt;font-family:&amp;quot;Times New Roman&amp;quot;;text-align:justify;text-justify:inter-ideograph;"&gt;&lt;!--Exclude empty div--&gt;&amp;#160;&lt;!--Exclude empty p--&gt;&lt;/p&gt;&lt;p style="margin-left:0pt;margin-right:0pt;margin-top:0pt;margin-bottom:.0001pt;font-size:10.0pt;font-family:&amp;quot;Times New Roman&amp;quot;;text-align:justify;text-justify:inter-ideograph;"&gt;The loss on investment in affiliate of $390,821 for the six months ended June 30, 2013, relates to the dilution effect from the Company&amp;#8217;s non-participation in the public offering of 4,000,000 common shares of Box Ships, which was completed on March 18, 2013.&lt;!--Exclude empty div--&gt;&lt;!--Exclude empty div--&gt;&lt;!--Exclude empty p--&gt;&lt;/p&gt;&lt;p style="margin-left:0pt;margin-right:0pt;margin-top:0pt;margin-bottom:.0001pt;font-size:10.0pt;font-family:&amp;quot;Times New Roman&amp;quot;;text-align:justify;text-justify:inter-ideograph;"&gt;&lt;!--Exclude empty div--&gt;&amp;#160;&lt;!--Exclude empty p--&gt;&lt;/p&gt;&lt;p style="margin-left:0pt;margin-right:0pt;margin-top:0pt;margin-bottom:.0001pt;font-size:10.0pt;font-family:&amp;quot;Times New Roman&amp;quot;;text-align:justify;text-justify:inter-ideograph;"&gt;As of December 31, 2012 and June 30, 2013, the Company held 3,437,500 shares or 16.4% and 13.8% of Box Ships&amp;#8217; common stock, respectively. Following such decrease, the Company has reassessed the accounting for the investment in Box Ships and concluded that the application of the equity method is still appropriate&lt;!--Exclude empty div--&gt;&lt;font color="#000000"&gt;.&lt;!--Exclude empty div--&gt;&lt;/font&gt;&lt;!--Exclude empty div--&gt;&lt;!--Exclude empty p--&gt;&lt;/p&gt;&lt;p style="margin-left:0pt;margin-right:0pt;margin-top:0pt;margin-bottom:.0001pt;font-size:10.0pt;font-family:&amp;quot;Times New Roman&amp;quot;;text-align:justify;text-justify:inter-ideograph;"&gt;&lt;!--Exclude empty div--&gt;&amp;#160;&lt;!--Exclude empty p--&gt;&lt;/p&gt;&lt;p style="margin-left:0pt;margin-right:0pt;margin-top:0pt;margin-bottom:.0001pt;font-size:10.0pt;font-family:&amp;quot;Times New Roman&amp;quot;;"&gt;Based on the closing price of Box Ships&amp;#8217; common share as of June 30, 2013, of $3.80, the fair value of the investment in Box Ships, which is considered to be determined through Level 1 inputs of the fair value hierarchy, was $13,062,500. As of June 30, 2013, the Company did not consider the difference between the fair value and the book value of the investment in Box Ships as other than temporary and therefore the investment was not impaired.&lt;!--Exclude empty div--&gt;&lt;!--Exclude empty p--&gt;&lt;/p&gt;&lt;/div&gt;</NonNumbericText><FootnoteIndexer /><CurrencyCode /><CurrencySymbol /><IsIndependantCurrency>false</IsIndependantCurrency><ShowCurrencySymbol>false</ShowCurrencySymbol><DisplayDateInUSFormat>false</DisplayDateInUSFormat></Cell></Cells><ElementDataType>nonnum:textBlockItemType</ElementDataType><SimpleDataType>na</SimpleDataType><ElementDefenition>Tabular disclosure of investments in and advances to affiliates.  Does not include the tabular disclosure of the disaggregation of investments in and advances to affiliates across legal entities.</ElementDefenition><ElementReferences>Reference 1: http://www.xbrl.org/2003/role/presentationRef

 -Publisher FASB

 -Name Accounting Standards Codification

 -Topic 946

 -SubTopic 320

 -Section S99

 -Paragraph 6

 -Subparagraph (SX 210.12-14)

 -URI http://asc.fasb.org/extlink&amp;oid=6955306&amp;loc=d3e611322-123010



Reference 2: http://www.xbrl.org/2003/role/presentationRef

 -Publisher SEC

 -Name Regulation S-X (SX)

 -Number 210

 -Section 14

 -Article 12



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