This study focused on the optimization of key process parameters for maximizing kefir grain biomass yield using statistical methodology. A response surface methodology (RSM) was developed to describe the effects of whey lactose and yeast concentrations, temperature and pH on increases in kefir grain biomass using cheese whey as a fermentation medium. Initially, one factor at a time was applied to evaluate the effect of different nitrogen sources. The results showed that the concentration of yeast extract significantly influenced the biomass increase. Then, a 2(4) full-factorial central composite design was used to optimize the process conditions. By using multiple regression analysis, the experimental data were fitted to a second-order polynomial model. RSM analysis indicated good correlation between experimental and predicted values. The most suitable combination of variables for higher biomass increase (76.13%) was 88.4 and 21.3 g/l, 5.2, and 20 A degrees C for concentration of whey lactose, concentration of yeast extract, pH, and temperature, respectively. At these optimal conditions, biomass increased by 81.34%, which was close to the amount predicted by the model.