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Summary: As current methods for antibiotic drug discovery are being outpaced by the rise of antimicrobial resistance, new methods and innovative technologies are necessary to replenish our dwindling arsenal of antimicrobial agents. To this end, we developed the PepSAVI‐MS pipeline to expedite the search for natural product bioactive peptides. Herein we demonstrate expansion of PepSAVI‐MS for the discovery of bacterial‐sourced bioactive peptides through identification of the bacteriocin Bac‐21 from Enterococcus faecalis pPD1. Minor pipeline modifications including implementation of bacteria‐infused agar diffusion assays and optional digestion of peptide libraries highlight the versatility and wide adaptability of the PepSAVI‐MS pipeline. Additionally, we have experimentally validated the primary protein sequence of the active, mature Bac‐21 peptide for the first time and have confirmed its identity with respect to primary sequence and post‐translational processing. Successful application of PepSAVI‐MS to bacterial secretomes as demonstrated herein establishes proof‐of‐principle for use in novel microbial bioactive peptide discovery. [ABSTRACT FROM AUTHOR]