Matrix metalloproteinases (MMPs) belong to a family of secreted or membrane-associated proteins capable of digesting extracellular matrix components. MMP-16 (MT3-MMP) is a member of the membrane-type MMP (MT-MMP) subfamily, which can be found in soluble form. MMP-16 activates pro-MMP-2 (gelatinase-A) that is involved in tumor development and metastasis, and is proposed as a therapeutic target for cancer. The SensoLyte® 520 MMP-16 Assay Kit uses a 5-FAM (fluorophore) and QXL™ 520 (quencher) labeled FRET peptide substrates for the continuous measurement of the enzyme activities. In an intact FRET peptide, the fluorescence of 5-FAM is quenched by QXL™ 520. Upon cleavage of the FRET peptide by MMP-16, the fluorescence of 5-FAM is recovered, and can be continuously monitored at excitation/emission = 490/520 nm. With superior fluorescence quantum yield and longer emission wavelength, 5-FAM/QXL™ 520 based FRET peptide is less interfered by the autofluorescence of test compounds and cellular components and provides better assay sensitivity. The assays are performed in a convenient 96-well microplate format.