Deoxynivalenol (DON) is a trichothecene mycotoxin that is produced by several species of Fusarium, which may infect grain crops. DON, as well as other type-B trichothecenes, contain an α,β-unsaturated carbonyl group that may react with sulfhydryl groups in, for example, amino acids and peptides. Such conjugates have been shown to occur in plants. Nucleophilic addition of thiols to the conjugated double bond in DON afforded several isomeric reaction products, and the thermodynamically favored isomers of DON-10-cysteine and DON-10-glutathione have been prepared and characterized previously. This study reports the preparation and characterization of the kinetically favored DON-10-cysteine isomer. We subsequently studied and compared the rate of the deconjugation reaction of the two DON-10-cysteine isomers and the thermodynamically favored DON-10-glutathione adduct. The deconjugation rate of the thermodynamically favored thiol conjugates was slow with half-lives of weeks even at pH 10.7, while the kinetically favored DON-10-cysteine isomer deconjugated within a few hours, affording free DON. We adapted a simple and rapid oxidation protocol in which the sulfide linkage was oxidized to a sulfoxide or sulfone that, when treated with the base, rapidly eliminated the adducted thiol as its sulfenate or sulfinate to afford free DON. The deconjugation reactions of the sulfoxides and sulfones of thermodynamically favored DON-10-thiols were complete within hours or minutes at pH 10.7, respectively. The increase in deconjugation rates for the kinetically favored DON-10-cysteine were less dramatic. Oxidation of sulfides to sulfoxides is known to occur in vivo, and thus, our data show that thiol-conjugated DON might become bioavailable via sulfide oxidation followed by elimination to regenerate DON. The oxidation-elimination approach could also be useful for the indirect quantification of DON-10-thiol conjugates in plant and animal tissues.