Instead of traditional methods, high-performance liquid chromatography can be used for the determination of enzymatic activities. Assays can be performed once the reaction mixture have been injected in the HPLC apparatus. The elution of the samples is carried out with suitable buffers, and the product(s) formed and left oven substrate(s) are quantitatively determined by means of an internal standard. HPLC assay is rapid, selective and comparable with traditional methods. Enzymatic activity can be easily measured in international units and the whole assay performed within 10–20 minutes. The results obtained for the urokinase assay are provided as a detailed example in the text