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 Homogenizer Method. The disrupted cells solution was centrifuged at 12,000 rpm for 20 min and the GSH content in the supernatant was analyzed by taking the optical density (OD) value of the solution using Spectrophotometer at 412 nm. Yeast solution with the concentration of 3, 6, 9 and 12 wt.% was disrupted at their original temperature for 30 sec to study the effect of yeast concentration. The result obtained shows that the optimum value of yeast concentration is 9 wt.% where 10.98 μmol mL-1 GSH was isolated. To study the effect of processing temperature, isolation process of the 9 wt.% yeast solution was done at various values of temperature such as 19, 22, 25, 28, 30 and 40°C for 30 sec. The results indicated that, the optimum GSH isolation temperature is 22°C with the isolation of 17.93 μmol mL-1 GSH. Then, to study the effect of isolation time, the isolation process of sample with the concentration of 9 wt.% was done at 22°C for 15, 30, 45 and 60 sec. The result indicated that the optimum isolation time for the Ultrasonic process is 30 sec.