N-terminomics is an approach that identifies N-terminal peptides originating from protease cleavage-sites (1). This technique has been instrumental for the investigation of the substrate structures cleaved by proteases. The protocol presented here describes how purified proteases can be applied to the E. coli proteome and screened for substrate cleavage-sites using N-terminomics and the companion software N-TerProt. This analysis can yield important amino acid specificity information about proteases, as well as their structural tolerance.