Exploring optimum and cost-efficient medium composition for microbial growth of Candida intermedia Y-1981 yeast culture growing on whey was studied by applying a multistep response surface methodology. In the first step, Plackett-Burman (PB) design was utilized to determine the most significant fermentation medium factors on microbial growth. The medium temperature, sodium chloride and lactose concentrations were determined as the most important factors. Subsequently, the optimum combinations of the selected factors were explored by steepest ascent (SA) and central composite design (CCD). The optimum values for lactose and sodium chloride concentrations and medium temperature were found to be 18.4g/L, 0.161g/L, and 32.4 degrees C, respectively. Experiments carried out at the optimum conditions revealed a maximum specific growth rate of 0.090 1/hr; 42% of total lactose removal was achieved in 24h of fermentation time. The obtained results were finally verified with batch reactor experiments carried out under the optimum conditions evaluated.