Abstract: The purpose of this study was to determine the effect of operating parameters such as the yeast concentration, processing temperature and isolation time on the isolation of GSH using Autolysis method. To study the effect of yeast concentration, yeast solution with the concentration of 15, 30, 45 and 60 wt.% was disrupted at their original temperature for 1 h. The result obtained shows that the optimum value of the concentration for the Autolysis was 15 wt.% with the yield of 0.3942 μmol mL-1 GSH. To study the effect of processing temperature, Autolysis process of the 15 wt.% yeast solution was done at a various values of temperature such as 15, 25, 28, 31, 33 and 40°C. The results indicated that, the optimum GSH isolation temperature (suitable processing temperature) is 28°C with the yield of 1.367 μmol mL-1 GSH. Then, to study the effect of isolation time, the Autolysis process of sample with the concentration of 15 wt.% was done at 28°C for 1, 2 and 3 h. The results indicated that the optimum isolation time for the Autolysis process is 1 h.