The optimum hydrolysis conditions of 2.5% (w/v) soy protein isolate (SPI) with 1% (w/w of SPI) Flavourzyme® for increasing glycerol release in mature 3T3-L1 adipocytes were investigated by response surface methodology (RSM). Higher glycerol release indicated higher lipolysis-stimulating activity. The independent variables were hydrolysis time (HT) 19.2 - 220.8 min, pH 5.32 - 8.68 and reaction temperature (RT) 33.2 - 66.8°C. Based on the response surface and contour plots, the optimum hydrolysis of SPI with Flavourzyme® for maximizing the glycerol release in the cells occurred at pH = 7.12, RT = 48.77°C and HT = 124.85 min. The F-value for lack of fit was not significant (p > 0.05), so the second order model was appropriate for describing the response surface. In addition, the model had a satisfactory coefficient of R2 (= 0.935) and was verified experimentally.