Abstract: Cell disruption techniques for intracellular product recovery operations result in the release of all cell constituents into surrounding medium, which complicates the downstream processing besides increase in the purification cost. However, these problems can be minimized using permeabilization technique. In the present investigation, the permeabilization of Kluyveromyces marxianus cells in relation to β-galactosidase (β-D-galactoside galactohydrolase, E.C. 3.2.1.23) activity was carried out using different organic solvents to avoid the problem of enzyme extraction. The permeabilization performance of the solvents was much dependent on their concentrations. Comparison of tested solvents has led to the selection of ethanol for maximal permeabilization of yeast cells. The optimum process conditions for permeabilization process were 50% (v/v) ethanol concentration, 25°C temperature and treatment time of 15 min. These permeabilized yeast cells displayed 90% lactose hydrolysis after 150 min of incubation time.