Cysteine disulfides, which constitute an important component in biological redox buffer systems, are highly reactive toward the hydroxyl radical (^•OH). The mechanistic details of this reaction, however, remain unclear, largely due to the difficulty in characterizing unstable reaction products. Herein, we have developed a combined approach involving mass spectrometry (MS) and theoretical calculations to investigate reactions of ^•OH with cysteine disulfides (Cys–S–S–R) in the gas phase. Four types of first-generation products were identified: protonated ions of the cysteine thiyl radical (⁺Cys–S^•), cysteine (⁺Cys–SH), cysteine sulfinyl radical (⁺Cys–SO^•), and cysteine sulfenic acid (⁺Cys–SOH). The relative reaction rates and product branching ratios responded sensitively to the electronic property of the R group, providing key evidence to deriving a two-step reaction mechanism. The first step involved ^•OH conducting a back-side attack on one of the sulfur atoms, forming sulfenic acid (–SOH) and thiyl radical (–S^•) product pairs. A subsequent H transfer step within the product complex was favored for protonated systems, generating sulfinyl radical (–SO^•) and thiol (–SH) products. Because sulfenic acid is a potent scavenger of peroxyl radicals, our results implied that cysteine disulfide can form two lines of defense against reactive oxygen species, one using the cysteine disulfide itself and the other using the sulfenic acid product of the conversion of cysteine disulfide. This aspect suggested that, in a nonpolar environment, cysteine disulfides might play a more active role in the antioxidant network than previously appreciated.