Abstract: The fragmentation mechanism of protonated 3-(phenylthio)-1H-indoles was studied by high-resolution electrospray ionization-high-energy collisional dissociation mass spectrometry (ESI-HCD-MS/MS), in combination with H/D exchange experiments and density functional theory (DFT) calculation. It was shown that under the mode of HCD of the protonated ion of 3-(phenylthio)-1H-indoles, free radical cation of 3-(substituted phenyl)-indole was obtained through phenyl nucleophilic substitution and rearrangement at C³ of the indole ring followed by elimination of mercapto free radical by homolytic cleavage of the C-S bond; indole free radical cation was obtained through 1,2-hydride shift followed by loss of thiophenyl radical by homolytic cleavage of the C³-S bond; and 3-thioxo-indole cation was obtained through 1,4-proton shift followed by loss of phenyl radical by charge-induced heterolytic cleavage of the C⁵-S bond.