Standard mechanism inhibitors are attractive design templates for engineering reversible serine protease inhibitors. When optimizing interactions between the inhibitor and target protease, many studies focus on the non-primed segment of the inhibitor's binding loop (encompassing the contact β-strand). However, there are currently few methods for screening residues on the primed segment. Here, we designed a synthetic inhibitor library (based on sunflower trypsin inhibitor-1) for characterizing the P2' specificity of various serine proteases. Screening the library against thirteen different proteases revealed unique P2' preferences for trypsin, chymotrypsin, matriptase, plasmin, thrombin, four kallikrein-related peptidases, and several clotting factors. Using this information to modify existing engineered inhibitors yielded new variants that showed considerably improved selectivity, reaching up to 7,000-fold selectivity over certain off-target proteases. Our study demonstrates the importance of the P2' residue in standard mechanism inhibition, and unveils a new approach for screening P2' substitutions that will benefit future inhibitor engineering studies.