A novel, generally applicable method of identifying peptides using HPLC, microwave-assisted acid hydrolysis (MAAH), and bioinformatics is described. Method validation was performed on bacteriocins—antibacterial peptides produced by probiotic bacteria—using nine different bacteriocin isolates secreted by the probiotic Lactococcus lactis. Calibration curves were constructed for 23 amino acid PTH derivatives, and analysis was performed using norleucine as the internal standard. Validation of amino acid analysis performed in the range 2.5–100 nmol/mL indicated excellent method linearity, while the LODs ranged from 0.17 to 2.88 nmol/mL and the LOQs from 0.51 to 8.75 nmol/mL. The MAAH method was developed by irradiating nisaplin for various durations at 700 W, with 7 min providing the best results. The amino acid content of each sample was estimated following the application of MAAH to ten different samples. The bacteriocins in our samples were identified using the UniProt database. Eight of nine peptides were identified as UniProt entries: nisin A (P13068), nisin Z (P29559), I4DSZ9, OB7236, P36499, OB7237, A0A0M7BH60, and T2C9F0. The phylogenetic tree was constructed for nisin A and nisin Z using the multiple sequence aligning tool Clustal Ω. The identified nisin types presented excellent correlation with their ModBase-predicted structures. The present method gives true, precise, and rapid results, and requires only standard technical equipment. Our results suggest that the present approach can facilitate the discovery of novel bacteriocins and provide useful information on not only the amino acid contents of peptides but also the evolution of protein biology. Graphical abstract Identification of eight bacteriocins secreted by the probiotic L. lactis, following microwave assisted acid hydrolysis (MAAH), amino acid content analysis of each sample with HPLC-DAD and bioinformatics analysis using Uniprot, Clustal Ω and ModBase.