Factor Xa (FXa) is a serine endopeptidase composed of two disulfide-linked subunits. FXa leads to blood clot formation by converting prothrombin to thrombin through the prothrombinase complex. FXa is generated from zymogen Factor X via the intrinsic and extrinsic pathways and is the rate-limiting step in the propagation of thrombin generation. In the presence of Ca2+ ions, FXa forms prothrombinase with factor Va on the phospholipid membrane of the activated platelets. FXa has emerged as an attractive target for drug discovery for thromboembolic diseases. The SensoLyte® 520 Factor Xa Assay Kit provides a convenient assay for high throughput screening of FXa inhibitors or continuous assay of enzyme activity. It detects FXa activity using a 5-FAM/QXL™ 520 fluorescence resonance energy transfer (FRET) peptide. In the intact FRET peptide, the fluorescence of 5-FAM is quenched by QXL™ 520. Upon cleavage into two separate fragments by FXa, the fluorescence of 5-FAM is recovered, and can be monitored at excitation/emission wavelengths = 490 nm/520 nm. With excellent fluorescence quantum yield and longer wavelength, 5-FAM shows less interference from autofluorescence of test compounds and cellular components. The assays are performed in a convenient 96-well microplate format.