Proteases have evolved to recognize a diverse range of polypeptide sequences. Remarkably, no protease is known that selectively cleaves its substrates adjacent to a site of post-translational modification. To explore the requirements for such a catalyst, we sought to identify mutations that would convert the bacterial protease subtilisin BPN', which preferentially cleaves peptides at phenylalanine or tyrosine residues, into a phosphotyrosine selective protease. Through iterative modeling, mutagenesis, and kinetic analysis, we have identified a subtilisin variant (E156R/P129G) that preferentially cleaves phosphotyrosine peptides relative to the optimal wildtype substrates. This double mutant achieves a 2500-fold enhancement in phosphotyrosine selectivity relative to the wild-type enzyme through the introduction of complementary steric and electrostatic features.