An exceptional MMP-2 (gelatinase A) and MMP-9 (gelatinase B) substrate this peptide is used for the continuous spectrophotometric assay of MMP-2 and MMP-9 in combination with a color-developing thiol-reactive agent, 4,4’-dithiodipyridine or Ellman’s Reagent (as indicators). This thiol peptide-enzyme reaction has a Km of 0.004 M and a kcat of 103 s-1. Using this substrate, collagenase can be detected at concentrations as low as 2 ng/mL. HPLC and tandem mass spectrometry are also used to analyze MMP-2 and MMP-9 in conjunction with this peptide substrate.