Many proteinases, including gelatinase B/MMP-9, fulfill crucial regulatory or effector functions in disease states and may be pharmacologically targeted by specific inhibitors. Denatured collagen type II provides one of the best gelatinase B substrates, and the characteristics of its cleavage were employed to define the requirements of a novel optimal substrate probe. A synthetic fluorescent derivative was used for the development of a new high-throughput technology for the selection of inhibitors on the principles of sensitivity of confocal fluorescence detection, resolution capacity of capillary electrophoresis, and multichannel power of DNA sequencers. Combinatorial chemical synthesis of a library of peptide-based inhibitors, library deconvolution, high-throughput screening, isolation, and mass spectrometric techniques enabled us to identify a novel single-peptide gelatinase B inhibitor. A notable finding is that the in vitro-selected inhibitor mimics many of the characteristics of the evolution-selected MMP propeptide sequence.