For mass plant growth-promoting inoculant production, a high-yield culture medium is fundamental. sequential application of statistical designs was used to optimize Azospirillum brasilense c16 biomass production. six nutritional (glycerol, glutamate, mannitol, citric acid, yeast extract and K2HPO4 3H2O) and three mineral sources (MgsO47H2O, Fecl3 and NaCl) were evaluated using five statistical experiments Placket-burman, factorial design, steepest ascent, response surface analysis, and mineral screening. the optimum medium composition (g L-1) was as follows: 28.33 glutamate, 2.92 yeast extract, 1.34 K2HPO4 3H2O, 0.5 MgsO47H2O and 0.02 Fecl3. After 24 hours of incubation, protein (32.04 μg) and dry biomass (1.51 g L-1) were 1.72 and 1.68 times higher than in conventional growth medium.