The specific effect of chemical and physical factors on amidase production from Geobacillus subterraneus RL-2a was investigated using design of experiments (DOE) methodology. The one-factor-at-a-time (OFAT) method was used to study the effects of carbon and nitrogen sources on amidase production. Subsequently, optimal levels of physical parameters and key media components, namely temperature, pH, sucrose, KHPO, NaCl, yeast, CaCl·2HO and MgSO·7HO, were determined using the Taguchi orthogonal array (OA) experimental design (DOE) methodology. Taguchi method based on three levels with a OA layout of L18 (2 × 3) with eight most influential factors on amidase synthesis for the proposed experimental design. Analysis of variance was performed on the obtained results and optimum condition suggested by statistical calculations was tested in a verification test. An increase of 169.56 % in amidase production compared to the unoptimized conditions was observed and the conversion of isonicotinamide was significantly improved after performing optimization techniques, including OFAT and Taguchi method. The result indicated that Taguchi method was effective in optimizing the culture conditions of amidase production.