Abstract: A method is disclosed for assaying a proteolytic enzyme comprising: (a) incubating an enzyme-containing sample with an immobilized fluorescence-quenched peptide having the formula Que-Sub-Flu-Spa-Car or Flu-Sub-Que-Spa-Car wherein Sub is a peptide chain containing a specific cleavage site for said proteolytic enzyme; Flu is a fluorophore; Que is a quencher capable of absorbing fluorescent radiation emitted by the fluorophore; Spa is a direct bond or a spacing chain; and Car is a water-insoluble and/or macromolecular carrier; (b) optionally separating the liquid from the carrier material; (c) irradiating said carrier material and measuring fluorescence. Also disclosed are immobilized substrates containing specific amino acid sequences for use in such an assay, especially in an assay for aggrecanase, and metalloproteinase-1, -3 and -13 activity.