gamma-Polyglutamic acid (gamma-PGA) is a biosynthetic outcome of glutamic acid polymerization by microbes. In the current study, we have isolated Bacillus methylotrophicus on solid differential media containing methylene blue. This is the first report mentioning the use of methylene blue to distinguish the monomeric and polymeric form of glutamic acid in the liquid medium using UV-Vis spectrophotometer. Our method can simplify the analytical process of gamma-PGA confirmation using the aforementioned studies. This screening protocol is sensitive to the detection of gamma-PGA quantities as low as 3 mu g/mL; thus, the potent producers can be effectively screened. Furthermore, we have carried out process optimization of the present strain for gamma-PGA production wherein we could obtain 1.4-fold improvement in the yield with respect to utilization of carbon source and 2.6-fold increase with respect to nitrogen source under submerged fermentation at a shake flask level. We have shown an increase in gamma-PGA titer from 1.5 to 36 g/L using mannitol, monosodium glutamate, peptone, and tween 20.