For synthetic applications of proteases, such as for peptide coupling, a combination of high esterase and low amidase activities is required. While achieving such specificity has been a long-standing goal, the decreases in amidase activity achieved to date have often also been accompanied by decreases in esterase activity. In the current study, a strategy of combined site-directed mutagenesis and chemical modification was applied to the serine protease subtilisin Bacillus lentus (SBL) to improve its esterase-over-amidase specificity. Using the crystal structure of SBL as a guide, the N62, L217, S166, and M222 active site residues were chosen for mutagenesis to cysteine and subsequent modification by alkyl methanethiosulfonate reagents. An initial rapid, combinatorial screen of the chemically modified mutant enzymes (CMMs) generated and, of their parent cysteine mutants, identified 25 promising candidates which were then subjected to detailed kinetic evaluations. Of these CMM and mutant enzymes, 20 exhibited an improvement, of up to 52-fold, in esterase-over-amidase activity compared to the wild type (WT). Furthermore, these increased esterase-to-amidase ratios were not gained at the expense of esterase activity, which was improved up to 3-fold higher than that of the WT in absolute terms. The general success of this approach is evident from the fact that, of the 25 CMMs and cysteine mutants evaluated, 19 displayed higher esterase activity than the WT, whereas only 3 had better than WT amidase activity.