Bacillithiol is a major low-molecular-weight thiol in gram-positive firmicutes including the human pathogen Staphylococcus aureus. Bacillithiol is regarded as an important defence mechanism against oxidants produced by the immune system, especially myeloperoxidase-derived hypochlorous acid (HOCl). However, it is unknown how fast BSH reacts with HOCl and what products are formed in the reaction. In the present study, we used sensitive MRM-based LC-MS methods to characterize the reaction of BSH with HOCl in cell-free solutions and in S. aureus. In the cell-free system, BSH formed predominantly the disulfide dimer (BSSB) at low mole ratios of HOCl and the sulfinic and sulfonic acids at higher oxidant concentrations. HOCl also promoted the formation of bacillithiol sulfonamide. In S. aureus, the oxidation pattern was similar except that a small proportion of BSH also formed mixed disulfides with protein thiols.Using competition with methionine, we determined the second-order rate constant for the reaction of HOCl with BSH to be 6 × 107 M-1s-1, which indicated a fast, near diffusion-controlled reaction. Other reactive halogen species, including hypothiocyanous acid (HOSCN), also produced bacillithiol sulfonamide, albeit to a smaller extent than HOCl. The sulfonamide was not produced by hydrogen peroxide, which instead formed BSSB.This study helps our understanding of BSH redox biology and provides tools for gauging the exposure of BSH-producing bacteria to oxidative stress.