Fragmentation reactions of protonated α-amino acids (AAs) were studied previously using tandem mass spectrometry (MS/MS) of unit mass resolution. Isobaric fragmentation products and minor fragmentation products could have been overlooked or misannotated. In the present study, we examined the fragmentation patterns of 19 AAs using high-resolution electrospray ionization MS/MS (HR-ESI-MS/MS) with collision-induced dissociation (CID). Isobaric fragmentation products from protonated Met and Trp were resolved and identified for the first time. Previously unreported fragmentation products from protonated Met, Cys, Gln, Arg, and Lys were observed. Additionally, the chemical identity of a fragmentation product from protonated Trp that was incorrectly annotated in previous investigations was corrected. All previously unreported fragmentation products and reactions were verified by pseudo MS3 experiments and/or MS/MS analyses of deuterated AAs. Clearer pictures of the fragmentation reactions for Met, Cys, Trp, Gln, Arg and Lys were obtained in the present study.