Optimization of the culture medium and process variables for xylitol production from corncob hemicelluloses hydrolysate was carried out using Candida guillermondii (NCIM 3124). The optimization was performed with statistical methodology based on experimental designs. The screening of nine nutrients for their influence on xylitol production was achieved using a Plackett-Burman design. The four selected components were optimized with Box-Behnken design using response surface methodology (RSM). The optimum level (g/l) is: MgSO4.7H2O- 1.34, yeast extract- 4.34, KH2PO4- 2.94 and xylose- 9.49 and influence of various process variables on the xylitol production was evaluated. The optimal levels were quantified by the central composite design using RSM. The optimum level of process variables are: temperature (29.88 oC), substrate concentration (3.26 g/l), pH (7.25), agitation speed (170.42 rpm), inoculum size (3.41 ml). These conditions were validated experimentally which revealed an enhanced xylitol yield of 0.73 g/g.