Knowledge on short-lived reaction intermediates is often essential for mechanistic investigations of organic reactions and for reaction optimization. Unfortunately, most conventional analytical methods are too slow to allow the detection of short-lived reaction intermediates. Herein, a direct laser desorption/ionization method coupled with linear quadrupole ion trap/orbitrap high-resolution tandem mass spectrometry was used for the detection and structural characterization of several previously proposed but undetected reaction intermediates formed during laser-induced UV/H2O2 advanced oxidation of 2-methylbenzoisothiazol-3-one. The elemental compositions of most detected (ionized) compounds were determined. Tandem mass spectrometry experiments based on gas-phase collision-activated dissociation (CAD) were conducted to gain information on the ion structures. The mechanisms of the CAD reactions were explored using high-level quantum chemical calculations to support the structures proposed for the neutral reaction intermediates formed during the laser-induced UV/H2O2 advanced oxidation of 2-methylbenzoisothiazol-3-one. In the negative-ion mode experiments, anions corresponding to three reaction intermediates were detected and structurally characterized: 1-hydroxy-2-methyl-1,2-dihydro-3H-1λ4-benzo[d]isothiazol-3-one, 2-(methylcarbamoyl)benzenesulfinic acid, and 2-(dihydroxy(oxo)-λ6-sulfaneyl)-N-methylbenzamide. One of the final products, 2-(methylcarbamoyl)benzenesulfonic acid, was also detected and characterized. In positive-ion mode experiments, cations corresponding to the reactant, 2-methylbenzoisothiazol-3-one, as well as an intermediate reaction product and the two final reaction products, 2-methylbenzo[d]isothiazol-3(2H)-one 1-oxide, N-methylsaccharine, and 2-(methylcarbamoyl)benzenesulfonic acid, respectively, were detected and identified. This research substantially improved the understanding on the reaction intermediates formed during laser-induced UV/H2O2 advanced oxidation of 2-methylbenzoisothiazol-3-one, which facilitates the delineation of the reaction mechanisms occurring in these processes.