The mechanism of proteolysis by serine proteases is a reasonably well-understood process. Typically, a histidine residue acting as a general base deprotonates the catalytic serine residue and the hydrolytic water molecule. We disclose here, the use of an unnatural d-amino acid as a strategic residue in P1 position, designed de novo based on the architecture of the protease catalytic site to impede the catalytic histidine residue at the stage of acyl-enzyme intermediate. Several probe molecules containing d-homoserine or its derivatives at P1 position are evaluated. Compounds 1, 6, and 8-10 produced up to 57% loss of activity against chymotrypsin. More potent and specific inhibitors could be designed with structure optimization as this strategy is completely general and can be used to design inhibitors against any serine or cysteine protease.