Recombinant hirudin, a potent and specific thrombin inhibitor, is considered for anticoagulant therapy. Therefore we developed a fast and sensitive chromogenic assay for its determination in plasma. Samples can be assayed directly if aprotinin, Polybrene® and urea are added to the reagent mixture. The influence of progressive inhibitors is excluded by a short incubation time. The samples (20 μl) are diluted with 1 ml reagent mixture (0.2 MTris, 0.025 MNaCl, pH 8.1, containing 0.833 M urea, 0.7 trypsin inhibitory units/ml aprotinin, 100 ng/ml Polybrene and 0.31 NIH units/ml bovine thrombin). After an incubation time of 1 min, 100 μl Chromoyzm TH® (Tos-Gly-Pro-Arg-pNA, 1.9 mM) is added. Δ absorbance/min is linear at least up to 3 min. The calibration curve is linear up to at least 800 ng hirudin/ml plasma. The inter- and intraassay coefficient of variation in hirudin spiked normal plasma is below 5%. The detection limit is at 25 ng hirudin/ml. In plasma samples obtained from healthy subjects under hirudin therapy, a good correlation was found to the activated partial thromboplastin time (r = 0.89). In conclusion, we describe a fast and simple chromogenic substrate test to assay hirudin in plasma. Under assay conditions, the influence of endogenous thrombin inhibitors can be neglected.