Enterokinase is a heterodimeric serine protease produced by cells in the duodenal wall. It converts trypsinogen to trypsin, indirectly activating a number of pancreatic digestive enzymes. Enterokinase is a key enzyme for intestinal digestion of proteins. Its deficiency may cause severe protein malabsorption. Enterokinase also plays an important role in acute experimental and clinical pancreatitis. The high degree of specificity exhibited by enterokinase for cleaving affinity or other tags from recombinant proteins makes it an ideal tool for use in biochemical applications. The SensoLyte® 520 Enterokinase Assay Kit is optimized for detecting activity of enterokinase. This kit employs a novel internally quenched 5-FAM/QXL FRET substrate for enterokinase. Enterokinase cleaves the FRET substrate into two separate fragments resulting in release of 5-FAM fluorescence which can be monitored at excitation/emission= 490/520 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 detect as low as 1.25 ng/mL of active enterokinase.