L- methioninase (methionine γ-lyase) has important biotechnological application because of its hydrolytic property to catalyze L- methionine to α- ketobutyrate, methaneethiol and ammonia. Nessler’s method was found to be simple and economical method to estimate L-methioninase activity in terms of released product ammonia. Response surface methodology was used as effective statistical tool to optimize conditions of enzyme because of accounting interactive effects of the variables with screening and prediction of large experimental domain. Process variables like temperature, pH, substrate volume and incubation period were considered as significant parameters for assay method of L-methioninase produced from Bacillus subtilis. RSM model has shown good fitness with value of coefficient of determination (R²) as 0.9864 for these assay parameters. Multiple regression analysis and ANOVA showed optimum assay conditions of pH of reaction mixture 8.5, reaction time of 30 minutes, incubation temperature of 37˚C, and substrate volumeof 0.5ml. The model was validated after performing experiments at these optimum assay parameters and activity of 17.4 Unit was estimated for L-methioninase.