A culture isolated from garden soil was found to be a promising L-glutaminase producer. Biochemical identification tests and 16S rRNA sequencing identified this isolate to be Klebsiella oxytoca. Subsequently, media optimization using one-factor-at-a-time approach and response surface methodology was undertaken. A face centered central composite design was employed to investigate the interactive effects of four variables, viz. concentrations of maltose, yeast extract, beef extract, and ammonium acetate on glutaminase production. Almost all factors had significant interactive effects on glutaminase production. A medium containing (g/L): maltose, 23.31; yeast extract, 20.0; beef extract, 20.01; ammonium acetate, 10.0; mannitol, 10.0; $KH_2PO_4$, 0.4; $Na_2SO_4$, 0.4; and $MgCl_2$, 0.4 was optimum for glutaminase production. The applied methodology was validated using this optimized media and enzyme activity of 458.91 ${pm}$ 9.49 U/L and specific activity of 0.441 ${pm}$ 0.04 U/mg protein after 42 h of incubation at $33^{circ}C$ were obtained.