To link cleaved substrates in complex systems with a specific protease, the protease active site specificity is required. Proteomic identification of cleavage sites (PICS) simultaneously determines both the prime-and non-prime-side specificities of individual proteases through identification of hundreds of individual cleavage sequences from biologically relevant, proteome-derived peptide libraries. PICS also identifies subsite cooperativity. To generate PICS peptide libraries, cellular proteomes are digested with a specific protease such as trypsin. Following protease inactivation, primary amines are protected. After incubation with a test protease, each prime-side cleavage fragment has a free newly formed N-terminus, which is biotinylated for affinity isolation and identification by liquid chromatography-tandem mass spectrometry. The corresponding non-prime sequences are derived bioinformatically. The step-by-step protocol also presents a web service for PICS data analysis, as well as introducing and validating PICS peptide libraries made from Escherichia coli.