Mechanism-based inhibitors (MBIs) are widely employed in chemistry, biology, and medicine because of their exquisite specificity and sustained duration of inhibition. Optimization of MBIs is complicated because of time-dependent inhibition resulting from multistep inactivation mechanisms. The global kinetic parameters kinact and KI have been used to characterize MBIs, but they provide far less information than is commonly assumed, as shown by derivation and simulation of these parameters. We illustrate an alternative and more rigorous approach for MBI characterization through determination of the individual microscopic rate constants. Kinetic analysis revealed the rate-limiting step of inactivation of the PLP-dependent enzyme BioA by dihydro-(1,4)-pyridone 1. This knowledge was subsequently applied to rationally design a second-generation inhibitor scaffold with a nearly optimal maximum inactivation rate (0.48 min-1).