Glutathione is a potent antioxidant that has shown promise in enhancing the processing of various foods and drinks such as bread and wine. Saccharomyces cerevisiae stands as a primary microorganism for glutathione production. This study sought to assess the potential of pulsed electric fields (PEFs) in extracting glutathione from S. cerevisiae cells. Yeast cells were subjected to PEF treatment (12 kV/cm, 150 μs) followed by incubation at varying pH values (4.0, 6.0, and 8.0) and temperatures (4 ◦C and 25 ◦C). Glutathione and protein extraction were assessed at different incubation times. Within one hour of incubation, PEF-treated yeast cells released over 60% of their total glutathione content, irrespective of pH and temperature. Notably, the antioxidant activity of the resulting extract surpassed that obtained through complete mechanical cell destruction and hot water, which form the conventional industrial extraction method in the glutathione industry. These results suggest that PEF could offer a rapid and more selective procedure, improving the extraction of this bioactive compound. Keywords: glutathione; pulsed electric fields; extraction; antioxidant; Saccharomyces cerevisiae