This study investigates the potential of additive-free extraction techniques to produce a proteolytically active yeast extract for use in the food industry. Brewer's spent yeast, a by-product of the brewing industry, is utilized as a feedstock, and thus a new route for its valorization is proposed. Four methods of releasing these components while maintaining their intrinsic bioactivity are investigated: thermal autolysis, ultrasonication, cell milling and high-pressure homogenization. Thermal yeast autolysis resulted in the highest release of protease activity, with 2.45 ± 0.05 U/g