Barley malting depends on hydrolytic enzymes that degrade storage macromolecules. Identifying barley cultivars with proteolytic activity that guarantees appropriate foaming, flavor, and aroma in the beer is of great importance. In this work, the proteolytic activity and profiles of brewing malt from Mexican barley cultivars were analyzed. Data showed that Cys‐ (at 50°C) and Ser‐proteases (at 70°C) are the major contributors to proteolytic activity during mashing. Essential amino acids, necessary for fermentation and production of good flavor and aroma in beer, were detected at the end of mashing. According to our results, Mexican cultivar HV2005‐19 exhibits similar proteolytic activities as those from cultivar Metcalfe, which is one of the most utilized for the brewing industry. Moreover, we propose Cys‐ and Ser‐proteases as biochemical markers during mashing at 50 and 70°C, respectively, to select barley cultivars for beer production. Practical applications Proteolytic activity, which depends on activation and de novo synthesis of proteases in the aleurone layer of barley seeds, is crucial in beer production. Identifying new barley varieties that have optimal proteolytic activities is of great interest for genetic improvement programs. In this study, we propose the variety HV2005‐19 as a genotype with Cys‐ and Ser‐proteases activity similar to that from Metcalfe, which is a top variety in the brewing industry.

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