Although proline is the most or second most abundant amino acid in wort and grape must, it is not fully consumed by the yeast Saccharomyces cerevisiae during alcoholic fermentation, unlike other amino acids. Our previous studies showed that arginine, the third most abundant amino acid in wort, inhibits the utilization of proline in most strains of S. cerevisiae. Furthermore, we found that some non-Saccharomyces yeasts utilized proline in a specific artificial medium with arginine and proline as the only nitrogen source, but these yeasts were not suitable for beer fermentation due to their low alcohol productivity. For yeasts to be useful for brewing, they need to utilize proline and produce alcohol during fermentation. In this study, 11 S. cerevisiae strains and 10 non-Saccharomyces yeast strains in the Phaff Yeast Culture Collection were identified that utilize proline effectively. Notably, two of these S. cerevisiae strains, UCDFST 40-144 and 68-44, utilize proline and produce sufficient alcohol in the beer fermentation model used. These strains have the potential to create distinctive beer products that are specifically alcoholic but with a reduction in proline in the finished beer.