Topical bovine thrombin is commonly used during surgery to maintain hemostasis and is rarely associated with abnormalities in hemostasis, including coagulopathies and bleeding. Coagulopathies may be related to the formation of cross-reactive antibodies to bovine factor V. Effectiveness of a new filtration step to remove factor V/Va from bovine thrombin was evaluated. A highly sensitive and specific Western blot capable of detecting minute amounts of factor V/Va and/or its fragments was developed. Samples were evaluated for bovine factor V related antigens using the Western blot method and a competitive enzyme-linked immunosorbent assay. Factor Va light chain fragment levels were detectable in crude thrombin and chromatographically purified thrombin but not in chromatographically purified and virally filtered preparations. Therefore, inclusion of the viral-filtration step during purification of thrombin is effective in reducing factor V or its fragments to undetectable levels, thus enhancing product purity.