This substrate has a fluorescent Trp residue in subsite P?2 and a dinitrophenol (Dnp) quenching group at the N-terminus. The quenching of the Trp fluorescence in the intact substrate is relieved upon hydrolysis of the P1-P?1 bond, giving rise to a continuously recording fluorescence assay. The residues placed in subsites P3-P?1 and P?3 is optimized for MMP-3, while Arg is placed in P?4 to enhance solubility. The substrate hydrolysis rate rivals or exceeds those of the corresponding natural protein substrates or other synthetic peptides. The solubility of this substrate in assay buffer exceeds the Km value for each reaction, allowing accurate determination of the kinetic parameters.