Abstract: Phenylsulfonylindoles are potentially useful precursors for anti-HIV and anti-inflammatory treatment. However, their fragmentation mechanistic studies in gas-phase chemistry have been seldom reported by now. In this paper, the fragmentation reactions for nine positional isomers of 3-phenylsulfonylindoles and 2-phenylsulfonylindoles were investigated comprehensively using negative ion mode electrospray ionization tandem mass spectrometry (ESI-MS/MS) with collisioninduced dissociation (CID). And density functional theory (DFT) calculation was used to compare the optimized ionic structures in their fragmentation processes. Due to the weak acidity of the nitrogen atom on the indole ring, the deprotonated ions of nine phenylsulfonylindoles were easily gained by eliminating the active hydrogen on the nitrogen atom under negative ion mode ESI, and the negative charge of deprotonated 3-phenylsulfonylindoles could localized at the N₁ or C₃ atoms, while the negative charge of deprotonated 2-phenylsulfonylindoles could localized at the N₁, C₂ or C₃ atoms. The characteristic neutral losses of SO₂ and phenyl radical were observed in the CID of nine deprotonated phenylsulfonylindoles. The electron withdrawing group on the benzene ring and electron-donating group on the indole ring facilitate the neutral loss of SO₂, while the electron-donating group on the benzene ring and electron-withdrawing group on the indole ring had the opposite effect. According to DFT-calculation, the neutral loss of SO₂ from deprotonated 3-phenylsulfonylindoles was initiated by an intramolecular nucleophilic aromatic attack of indole anion to the C₈ atom of aryl ring through a three-membered ring transition accompanied with heterolytic cleavage of C₈-S bond and C₃-S bond. In the case of deprotonated 2-phenylsulfonylindoles, the elimination of SO₂ was gained through a two-step nucleophilic aromatic substitution of indole anion companied with heterolytic cleavage of C-S bond: the N₁ atom of indole anion nucleophilically attacked the C₈ atom of aryl ring by a four-membered ring transition, then the C₂ atom of indole anion nucleophilically attacked the C₈ atom of aryl ring through a three-membered ring transition. The homolytic cleavage of C₈-S bond of deprotonated phenylsulfonylindoles resulted in the neutral loss of phenyl radical. Attributed to the different shift process of the indole anion, the abundance ratio of R₁R₂C₁₄H₈N^- resulted from the neutral loss of SO₂ to the R₁C₈H₄SO₂N^-• originated from phenyl radical loss is less than 1 in the fragmentation of deprotonated 3-phenylsulfonylindoles, while this ratio is much more than 1 for deprotonated 2-phenylsulfonylindoles. It is obvious that the above-mentioned two positional isomers can be distinguished quickly, easily and accurately by ESI-MS/MS in negative ion mode based on the difference in the ratio of R₁R₂C₁₄H₈N^- to the R₁C₈H₄SO₂N^-•. This study can provide a theoretical basis for the structural identification and quantitative analysis of phenylsulfonylindoles.