Abstract An amidolytic assay was developed for measuring factor V in human plasma. Diluted plasma is incubated at 37°C with lipid, Ca ++, Taipan Snake Venom (Oxyuranus Scutellatus Scutellatus) and bovine prothrombin. The reaction is stopped after 4 min with EDTA. The content of factor V in samples is then determined by measuring the amidolytic activity of formed thrombin on synthetic chromogenic peptide substrate S-2238, H-D-Phe-Pip-Arg-pNA. The assay appears to be specific for factor V as: 1) human congenitally deficient FV plasma gives no activity in the system, 2) deficiency of factor II, VII, VIII, IX, X, XI or XII does not affect the determination of FV and 3) no prothrombin is activated if the assay is run without venom. The correlation coefficient r between this amidolytic assay and a clotting assay was 0.91 (n=20). The intra-assay coefficient of variation was 3.7% for the amidolytic method.