Media composition for Cephalosporin-C acylase (CPC acylase) was optimized using a stepwise strategy. Out of the fifteen nutrients, five key ingredients viz. glucose, beef extract, glutamic acid, adipic acid and sodium chloride were identified for best medium composition by applying Plackett-Burman (PB) statistical design. Results indicated that above factors negatively affects the CPC acylase production i.e. increase in the concentration of these factors decreases the acylase production. The optimal concentration of key ingredients was determined using Central Composite Design (CCD) and the results obtained were fitted with a second order polynomial model. By applying the optimized conditions, a model was designed which predicted the CPC acylase activity as 9.56 U/mL which was in close resemblance to the experimental value of 11.2 U/mL.