Abstract: The enzyme β-galactosidase, have been used in the dairy industry for the improvement of lactose intolerance. However, the industrial applications of enzymatic hydrolysis processes are being hampered, due to intracellular location of the yeast enzyme. The treatment of yeast cells with various chemical agents (n-butanol, n-propanol, iso-propanol, acetone, ethanol, toluene and a mixture of organic solvent) have been carried out to increase the β-galactosidase activity. Response Surfaces Methodology (RSM) using Central Composite Rotable Design (CCRD) was employed to optimize the toluene (25%, v/v): ethanol (50%, v/v) ratio, treatment time and temperature. The optimum operating conditions for permeabilization process to achieve maximum β-galactosidase activity obtained by RSM were 1:1 ratio of toluene (25%, v/v) and ethanol (50%, v/v), 23.0°C temperature and treatment time of 12 min which displayed enzyme activity of 1.68 IU mg-1 DW. The use of permeabilized cells can help to overcome the problems/costs associated with enzyme extraction and purification from yeast cells and in the development of a low-cost technology for β-galactosidase production.