Saccharomyces cerevisiae PTCC5269 growth was evaluated to specify an optimum culture medium to reach the highest protein production. Experiment design was conducted using a fraction of the full factorial methodology, and signal to noise ratio was used for results analysis. Maximum cell of 8.84 log (CFU/mL) was resulted using optimized culture composed of 0.3, 0.15, 1, and 50 g L-1 of ammonium sulfate, iron sulfate, glycine, and glucose, respectively at 300 rpm and 35 A degrees C. Glycine concentration (39.32 % contribution) and glucose concentration (36.15 % contribution) were determined as the most effective factors on the biomass production, while Saccharomyces cerevisiae growth had showed the least dependence on ammonium sulfate (5.2 % contribution) and iron sulfate (19.28 % contribution). The most interaction was diagnosed between ammonium sulfate and iron sulfate concentrations with interaction severity index of 50.71 %, while the less one recorded for glycine and glucose concentration was equal to 8.12 %. An acceptable consistency of 84.26 % was obtained between optimum theoretical cell numbers determined by software of 8.91 log (CFU/mL), and experimentally measured one at optimal condition confirms the suitability of the applied method. High protein content of 44.6 % using optimum culture suggests that Saccharomyces cerevisiae is a good commercial case for single cell protein production.