Bacterial proteases play an important role in a broad spectrum of processes, including colonization, proliferation and virulence. In this respect, bacterial proteases are potential biomarkers for bacterial diagnosis and targets for novel therapeutic protease inhibitors. To investigate these potential functions, the authors designed and used a protease substrate fluorescence resonance energy transfer (FRET)-library comprising 115 short D- and L-amino-acid-containing fluorogenic substrates as a tool to generate proteolytic profiles for a wide range of bacteria. Bacterial specificity of the D-amino acid substrates was confirmed using enzymes isolated from both eukaryotic and prokaryotic organisms. Interestingly, bacterial proteases that are known to be involved in housekeeping and nutrition, but not in virulence, were able to degrade substrates in which a D-amino acid was present. Using our FRET peptide library and culture supernatants from a total of 60 different bacterial species revealed novel, bacteria-specific, proteolytic profiles. Although in-species variation was observed for Pseudomonas aeruginosa, Porphyromonas gingivalis and Staphylococcus aureus. Overall, the specific characteristic of our substrate peptide library makes it a rapid tool to high-throughput screen for novel substrates to detect bacterial proteolytic activity.