Thrombin, a serine protease, plays a central role in hemostasis by converting soluble plasma fibrinogen into an insoluble fibrin clot and by promoting platelet aggregation. Thrombin also influences a number of normal and pathological processes, including inflammation, tissue repair, embryogenesis, angiogenesis, and tumor invasion. The SensoLyte® 520 Thrombin Assay Kit is optimized for detecting thrombin activity. This kit contains a novel internally quenched 5-FAM/QXL™ 520 FRET substrate for thrombin. Thrombin cleaves the FRET substrate into two separate fragments resulting in the release of 5-FAM fluorescence which can be monitored at excitation/emission= 490 nm/520 nm. The long wavelength fluorescence of 5-FAM is less interfered by the autofluorescence of components in biological samples and test compounds. The assay can be performed in a convenient 96-well or 384-well microplate format for screening of enzyme inhibitors.