In this study, we use a combination of tandem mass spectrometry and hydrogen radical-mediated fragmentation techniques to analyze the sequence of peptides. We focus on fragmentation induced by the attachment of hydrogen atoms to the histidine and tryptophan residue side-chains in the peptide that occurs in the gas-phase. The hydrogen atom attached to the imidazole and indole rings in the histidine and tryptophan residues, respectively, and the resulting intermediate experienced Cα-Cβ bond cleavage. The detailed fragmentation mechanism is investigated by computational analysis using density functional theory. According to the results, hydrogen attachment occurs at the C-5 position in histidine and at the C-2 position in the tryptophan, which has a lower activation energy compared with the other positions and the resulting intermediate radicals yielded fragments due to Cα-Cβ bond cleavage. For the peptides that contain the histidine and tryptophan residues, cleavages in the Cα-Cβ and N-Cα bonds occurred independently. Therefore, the method presented in this study is applicable when analyzing peptides that contain histidine and tryptophan residues.