Urokinase Plasminogen Activator (uPA), also known as u-plasminogen activator or urokinase, is a highly-specific serine protease from the peptidase S1 family that cleaves plasminogen to form plasmin making it a key player in the plasminogen activator (PA) system. Expression of uPA is minimal in normal cells but is increased several fold in tumor cells by extracellular stimuli elevated in cancer and corresponds to poor outcomes in several types of cancer. Therefore, uPA has been identified as an excellent target for therapeutic development through inhibition of protease activity or though inhibition of uPA-dependent signaling while in complex with uPA receptor (uPAR). The activity assay of uPA was measured by its ability to cleave a peptide substrate, N-carbobenzyloxy-Gly-Gly-Arg-7-amido-4-methylcoumarin (Z-GGR-AMC). The reaction was performed in 50 mM Tris, 0.01% Tween-20, pH 8.5 ( Assay Buffer), ainitiated by addition 50 μL of 1.5 ug/ml uPA (diluted by Assay Buffer) to 50 µL of 200 uM Substrate. Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively, in kinetic mode for 5 minutes. The specific activity of recombinant mouse uPA is >2000 pmol/min/µg.