Using expressed protein ligation, five unnatural tyrosine analogues (amino-phenylalanine, homotyrosine, 2-methyl-tyrosine, (alphaS,betaR)-beta -methyl-tyrosine, and 2,6-difluoro-tyrosine) were incorporated into Src in place of the natural tail tyrosine residue. These semisynthetic substrates were evaluated as Csk substrates or allosteric activators. It appears that the tyrosine phenol hydroxyl is unlikely to be contributing significantly to Src's ground-state binding affinity for Csk. It has been observed that stabilizing tyrosine conformers can further optimize Src's already high substrate efficiency. These latter findings contrast similar studies with synthetic peptide substrates and highlight the value of investigation of protein kinase substrate selectivity with protein substrates.