ABSTRACT The objective of the present study was to develop a methodology to obtain the enriched fraction of ram seminal vesicle protein 14 (RSVP14). The study was developed using Morada Nova rams, from which semen samples were collected weekly. Seminal plasma proteins were precipitated with cold ethanol, and then 6.15 mg/mL of total proteins were subjected to liquid gelatin affinity chromatography using a Gelatin-Sepharose matrix coupled to an automated chromatographic system. Proteins were eluted into four fractions (A, B, C, and D), in which A and B contained non-gelatin-binding proteins, and C and D fractions contained gelatin-binding proteins. Gels were analyzed by Quantity One software, in which five protein bands were detected in fraction D, with molecular weights between 12 and 30 kDa. The gelatin-binding proteins (fraction D) were loaded into a HiTrap™ Heparin HP affinity column. Two chromatographic fractions were separated (D1 and D2), in which D1 contained non-heparin-binding proteins, and D2 contained heparin-binding proteins. Proteins from the last two peaks were subjected to 12.5% SDS-PAGE and Western Blot. Two bands with molecular weight of 14 and 24 kDa, contained in fraction D1, were excised from gel and subjected to tandem mass spectrometry, identifying the proteins RSVP14 and RSVP24. Thus, the chromatographic methods of the present study are efficient to capture the enriched fraction of RSVP14.