Matrix metalloproteinases (MMPs) belong to a family of secreted or membrane-associated proteins capable of digesting extracellular matrix components. The importance of MMPs in tumor development and invasion and other diseases is well known. MMP-3 (stromelysin-1, transin-1) is proposed as a potential therapeutic target. SensoLyte® 520 MMP-3 Assay Kit uses a 5-FAM (fluorophore) and QXL520™ (quencher) labeled FRET peptide substrates for continuous measurement of the enzyme activities. This substrate showed excellent specificity to MMP-3 with minimal cross-reaction with MMP-1, 2, 7, 8, 9, 13 and 14. In an intact FRET peptide, the fluorescence of 5-FAM is quenched by QXL520™. Upon the cleavage of the FRET peptide by MMP-3, the fluorescence of 5-FAM is recovered, and can be continuously monitored at excitation/emission = 490 nm/520 nm. With superior fluorescence quantum yield and longer emission wavelength, 5-FAM/QXL520™ 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 or 384-well microplate format.