A HPLC-MS(MS) method is presented here for reaction monitoring of the oxidation of isothiazolium salts with H2O2/acetic acid. The comprehension of this mechanism was the aim of this work using several N-functionalized substances for the investigation. The strong acidic reaction mixture was separated on a RP-18 reversed-phased column without any sample pretreatment and occurring intermediates were identified by API-MS(MS) techniques. Thus, it was possible to gain detailed information about the course of oxidation and to develop an oxidation scheme of existing reaction pathways.