Methodsa method for identifying the cleavage sites for proteases in complex proteomes is described. The method makes use of a computer program called MS‐Cleave. The utility of the approach is demonstrated by determining of the cleavage sites in a set of recombinant Escherichia coli proteins generated by two matrix metalloproteinases (MMPs), MMP‐9 and MMP25. MS‐Cleave analyzes LC‐MS/MS data of standard samples with no additional chemical labels, processes them through a stringent peptide identification procedure, filters out tryptic cleavages and terminal trimming products. Each cleavage site is given a score based on the cleavage efficiency.ResultsTwenty‐two E. coli proteins were subjected to proteolysis by either MMP‐9 or MMP‐25. The cleaved samples were combined and analyzed by LC‐MS/MS. The cleavage sites are validated by comparison with the known substrate specificity of the two proteases which were defined with a substrate phage display library.ConclusionMS‐Cleave analysis is a reliable and convenient approach for the identification of protease cleavage sites without the use of additional chemical tags. The method is applicable for any standard proteomics facility.This work was supported by NIH 5U54RR020843‐05 grant to JWS, NIH 5R01RR016522‐05 and 1‐P41‐RR024851‐01 grants and Howard Hughes Medical Institute Professor Award to PP.