In contrast to earlier assays, the prothrombin determination described here uses a two-step reaction: first, prothrombin is activated with human factor Xa, phospholipids, Ca++ and factor V; second, the amount of activated prothrombin is assayed by the proteolysis of added fibrinogen. The assay is dependent only on prothrombin, and independent of other components of thrombin generation. Carboxy- and decarboxyprothrombin can be differentiated by means of using Echis carinatus venom instead of factor Xa as activator. The micro-coagulation assay is compared with a conventional one-stage coagulation test, with a prothrombin determination using chromogenic substrate and immunologically by Laurell electrophoresis.