An assay for human plasminogen using NU-CBZ-L-lysine p-nitrophenyl ester as the substrate is described. The procedure is performed by activating the sample with streptokinase for 5 min at pH 6.0 and 25", the substrate is added to this mixture and its hydrolysis is monitored spectrophotometrically at 340 nm for up to 4 min. The initial rate of the product formation is proportional with enzyme concentration from IO to 300 p&ml. The rate is independent of streptokinase concentration in the range used in the assay. The antifibrinolytic agent 6-aminohexanoic acid interferes with the assay. A standard plasminogen preparation from the Committee on Thrombolytic Agents (CTA) of the National Heart Institute was assayed to determine the conversion factor for the two methods. This factor is 2.0 CTA units per unit in the new assay. These data show that this assay is more specific and convenient than other methods using N"-tosyl-L-arginine methyl ester or N"-benzoyl-L-arginine ethyl ester as substrates. The method was also applied to measuring plasminogen in plasma. Since the endogeneous plasma inhibitors did not interfere in this method. the assay was performed on plasma directly. The assay requires only 100 ~1 of plasma and takes less than I5 min.