Disclosed is a method for evaluating enzyme inhibitory activity of a known or putative inhibitor or modulator of an enzyme. The enzymes under investigation are those having a low-barrier hydrogen bond present in an active site of the enzyme. The compound whose activity is to be evaluated is contacted to an enzyme having a low-barrier hydrogen bond present in an active site of the enzyme. The presence, absence, or electronic character of the low-barrier hydrogen bond is then measured. The method is useful for designing mechanistic-based inhibitors or modulators of aspartic proteases and other enzymes.