In this study, cultures of Lactobacillus acidophilus was used as the source of intracellular Beta-galactosidase. Release of this enzyme from the bacterial cells is a decisional step for its recovery and further downstream processing. This study compares some methods for cell disruption, such as ultrasound, lysis by lysozyme and combination of lysozyme and ultrasound. Response surface methodology (RSM) was used to optimize the conditions for cell disruption. The various means of disruption were compared by hydrolytic activity Beta-glycoside linkages of enzyme Beta-galactosidase. The Beta-galactosidase activity was measured using Alpha-nitrophenyl- Beta-D-galactopyranoside (ONPG). The results indicated that combination of ultrasound and lysozyme is the best method for L acidophilus cell disruption. The crude extract had Beta-galactosidase activity 893.61 + or - 9.24 U from 1 g dry cell weight.