A theory and experimental method are presented to characterize the kinetics of fast-acting, irreversible proteinase inhibitors. The theory is based upon formal analysis of the case of an irreversible inhibitor competing with a substrate for the active-site of a proteinase. From this theory, an experimental method is described by which the individual microscopic kinetic constants for the interaction of the inhibitor with the proteinase can be determined. These are, for a two-step inhibition reaction sequence, the equilibrium dissociation constant and the first-order rate constant for inhibition, and, for a one-step inhibition reaction sequence, the second-order rate constant for inhibition. The theory and experimental method were validated by an analysis of the inhibition of trypsin by the two-step synthetic inhibitor p-nitrophenyl p-guanidinobenzoate and the one-step protein inhibitor bovine pancreatic trypsin inhibitor. The substrate used in these experiments is a new, fluorogenic substrate for trypsin-like serine proteinases (Cbz-Ile-Pro-Arg-NH)2-Rhodamine, the synthesis and properties of which are described.