L-N-Acetyl-O-(arylalkyl)-serines incorporating two stereocenters were prepared via ring-opening reactions of an aziridine precursor, derived from L-serine, with each of R and S 1-phenyl- and 2-methoxy-2-phenylethanol. These were then evaluated as inhibitors of the serine proteases subtilisin Carlsberg and α-chymotrypsin in order to probe the effects on binding of the different stereocenter configurations. Each L-N-Ac-O-(arylalkyl)-serine derivative was a competitive inhibitor for both enzymes, but significant stereocenter-configuration discrimination was observed only in the inhibition of subtilisin Carlsberg by the enantiomeric O-(1-phenylethyl)-serine derivatives.