l-Methionine γ-lyase (EC 4.4.1.11) is a pyridoxal 5 ' -phosphate-dependent multifunctional enzyme. Measuring the initial velocity of α-ketobutyrate production by α,γ-elimination of l-methionine catalyzed by l-methionine γ-lyase is not very feasible, because the enzyme simultaneously catalyzes both γ-replacement and α,γ-elimination. To develop an accurate enzyme assay, the comprehensive enzyme kinetics needed to be elucidated by progress curve analysis on the basis of a reaction model for conversion of l-methionine to α-ketobutyrate, methanethiol, and ammonia with pyridoxal 5 ' -phosphate as a cofactor. Kinetic parameters were determined by linear transformation using an approximation of a Maclaurin series from the whole velocity of α-ketobutyrate production including α,γ-elimination and γ-replacement. The significance of γ-replacement was revealed both theoretically and practically by the kinetic analysis. The enzyme activity was standardized and represented as the V m a x value taking into consideration γ-replacement in the presence of l-methionine at 37 o C and pH 8.0. The novel method that we proposed is accurate, sensitive, reproducible, and linear over a wide range for the determination of l-methionine γ-lyase activity.