Sulfur mustard (SM), a vesicant chemical warfare agent, is a strong alkylating compound that readily reacts with numerous biomolecules. The goal of the present work was to define and validate new biomarkers of exposure to SM. Because investigations using SM are prohibited by the Organization for the Prohibition of Chemical Weapons, we worked with 2-chloroethyl ethyl sulfide (CEES), a monofunctional analog of SM. The studied biomarkers are the conjugate of CEES to glutathione (GSH) and two of its metabolites, the cysteine (Cys) and the N-acetyl-cysteine (NAC) conjugates. After synthesizing the specific biomarkers, a UHPLC-MS/MS method with on-line solid phase extraction and isotopic dilution was optimized. The method was first applied to mice cutaneously exposed to CEES. Emphasis was placed on the systemic diffusion of the vesicant (Fig. 1). This was shown first by the detection of the three conjugates in plasma. A plasma metabolomics study confirmed the strong systemic impact following a self-protection mechanism to alleviate the injury of CEES exposure. Because neurological disorders have been reported in SM-exposed humans, we then studied the consequences of exposure to CEES in brain. The N7Gua-CEES adduct could be detected in nuclear DNA. Similarly, GSH-CEES, Cys-CEES and NAC-CEES were present in brain extracts until two weeks after exposure. Altogether, these results, based on novel exposure markers, confirm the ability of vesicants to induce internal damage following dermal exposure. The observation of alkylation damage to glutathione and DNA in brain provided mechanistic information of the neurological insult of vesicants. The method was recently extended to biomarkers of sulphur mustard. Work on SM-exposure is ongoing in mice topically exposed to SM and rabbits contaminated in the eyes. [1].M. Roser, D. Beal, C. Eldin, L. Gudimard, F. Caffin, F. Gros-Desormeaux, D. Leonco, F. Fenaille, C. Junot,, C. Pierard, C. T. Douki, (2021) Anal. Bioanal. Chem. 413, 1337–1351. [2].M. Gilardoni, D. Leonco, F. Caffin, F. Gros-Desormeaux,, C. Eldin, D. Beal, S. Ouzia, C. Junot, F. Fenaille, C. Pierard, C., T. Douki, T. (2021) Toxicology 462, 152950.