Hirudin is a potent and specific thrombin inhibitor. Since recombinant hirudin is being considered for anticoagulant and antithrombotic therapy we developed a fast and sensitive chromogenic substrate assay for its determination in plasma. The plasma samples (20 μl) were incubated with 1 ml reagent mixture (0.2 M Tris buffer, 0.025 M NaCl, pH 8.1, containing 0.833 M urea, 0.7 trypsin inhibitor U/ml aprotinin, 100 ng/ml Polybrene and 0.31 NIH U/ml bovine thrombin) for 1 min. Thereafter 100 /d Chromozym TH (Tos-Gly-Pro-Arg-pNA, 1.9 mM) was added. The change in absorbance/min (AA/min) was recorded at 405 run. AA/min was linear for at least 3 rain. The calibration curve was linear at least up to 800 ng hirudin/ml plasma. Intra-assay and inter-assay coefficients of variation were 2.8–31% and 5.3–5.8% respectively. The influence of progressive thrombin inhibitors can be neglected because of the short incubation time. Plasma samples can be assayed directly if aprotinin, polybrene and urea are added to the reagent mixture.