Matrix‐assisted laser desorption/ionization in‐source decay (MALDI‐ISD) causes the selective cleavage of Cα–C peptide bonds when an oxidizing matrix is used, and the fragmentation involves the hydrogen abstraction from a peptide by a matrix. The hydrogen abstraction from either an amide nitrogen or β‐carbon atom has been proposed to be the initial step leading to the Cα–C bond cleavage. In this regard, the production of [a]+ fragments originated upon bond cleavage at the C‐terminal side of phenylglycine residues strongly suggested that that the Cα–C bond cleavage occurred through a nitrogen‐centered radical intermediate and that the fragmentation through a β‐carbon‐centered radical intermediate can be ruled out from the MALDI‐ISD process, because phenylglycine residues do not contain β‐carbon atoms. The Cα–C bond cleavage of such nitrogen‐centered radical initially produced an [a]•/[x − H] fragment pair, and then the [a]• radical either reacted with the matrix or underwent loss of the side‐chain, leading to [a − H] or [d − H] fragment. The Cα–C bond cleavage at the C‐terminal side of phenylglycine and phenylalanine residues only generated [a]+ fragments, whereas that of homophenylalanine and S ‐methylated cysteine residues provided both [a]+ and [d]+ fragments. The yield of [d]+ fragments was dependent on the chemical stability of the resultant radicals formed upon side‐chain loss. MALDI‐ISD produced [M − H + matrix]+, [M − 16 + H]+, [M − 32 + H]+, and [d]+ fragments, when the analyte peptide contained a methionine residue. These fragments were formed upon abstraction of a hydrogen atom from the side‐chain of a methionine residue and its subsequent reaction with the matrix. The oxidation of methionine residues suppressed the hydrogen abstraction from their side‐chain.