Sulfur dioxide (SO2) is widely used in winemaking for its antioxidant and antimicrobial properties, but its application is increasingly questioned due to health concerns and regulatory restrictions. Glutathione (GSH) has emerged as a promising alternative, effectively preventing oxidative browning and preserving key aroma compounds. This study presents an integrated approach to enhance GSH production and extraction from Saccharomyces cerevisiae, combining amino acid supplementation and Pulsed Electric Fields (PEF) technology. Optimal supplementation with glutamic acid, cysteine, and glycine at 30 h increased intracellular GSH accumulation by 53 % (yielding 443 ± 7.9 mg GSH /L of culture). PEF treatment (20 kV/cm, 150 μs, 153.3 kJ/kg) achieved >90 % membrane permeabilization, allowing the recovery of 66 % of intracellular GSH. The lyophilized extract contained 51.2 ± 1.32 mg GSH/g, with a GSH/GSSG ratio of 19.2, exceeding the OIV standard for inactivated yeast preparations. The PEF-GSH extract exhibited higher antioxidant capacity than a commercial inactivated yeast product in aqueous assays, nonetheless, both showed comparable efficacy in mitigating oxidative browning in grape must. The combination of GSH-derived extracts with SO2 enabled a 50 % reduction in required sulfite concentration while maintaining must stability. Additionally, the PEF-GSH extract exhibited lower high-molecular-weight protein content, minimizing turbidity risk and enhancing its post-fermentation applicability. These findings support PEF-assisted extraction as an efficient strategy for producing GSH-rich extracts, offering a viable alternative to reduce SO2 use in winemaking.