A method has been developed for accurately and precisely measuring the activity of a range of plasminogen activators (PAs) used as thrombolytic agents, including streptokinase, tissue plasminogen activator (tPA) and variants, and urokinase (uPA), both single and two chain forms. Plasminogen activation is monitored in a transparent, solid fibrin matrix but uses chromogenic substrate hydrolysis, rather than changes in fibrin, to quantitate the activity of PAs. The method has been tested in two recent international collaborative studies involving tPA and streptokinase where it has been shown to perform very well. Furthermore, the method is based on sound enzymological principles and once correction for the competitive inhibition of fibrin(ogen) is made, the generation of plasmin can be determined in molar terms and hence the activity of PAs can be expressed and compared in SI units (rate of increase in molar concentration of plasmin) as well as International Units. The assay is also arranged in such a way to reflect the behavior of PAs in vivo during thrombolytic therapy and it is shown that the specific activity of streptokinase and tPA in this system reflects plasmin generation capacity of these thrombolytics for doses given in infusions for treatment of myocardial infarction. The method would make a suitable reference method for PAs and provides a rigorous means of studying and modeling the enzymology of fibrinolysis and will be helpful in the rational design of third generation thrombolytic agents.