Ras GTPases cycle between active GTP-bound and inactive GDP-bound forms to regulate a multitude of cellular processes, including cell growth, differentiation, and apoptosis. The activation state of Ras is regulated by protein modulatory agents that accelerate the slow intrinsic rates of GDP dissociation and GTP hydrolysis. Similar to the action of guanine-nucleotide exchange factors, the rate of GDP dissociation can be greatly enhanced by the reaction of Ras with small-molecule redox agents, such as nitrogen dioxide, which can promote Ras activation. Nitrogen dioxide is an autoxidation product of nitric oxide and can react with an accessible cysteine of Ras to cause oxidation of the bound guanine nucleotide to facilitate Ras guanine nucleotide dissociation. Glutathione has also been reported to modify Ras and alter its activity. To elucidate the mechanism by which glutathione alters Ras guanine nucleotide binding properties, we performed NMR, top-down and bottom-up mass spectrometry, and biochemical analyses of glutathiolated Ras. We determined that treatment of H-Ras, lacking the nonconserved hypervariable region, with oxidized glutathione results in glutathiolation specifically at cysteine 118. However, glutathiolation does not alter Ras structure or biochemical properties. Rather, changes in guanine nucleotide binding properties and Ras activity occur upon exposure of Ras to free radicals, presumably through the generation of a cysteine 118 thiyl radical. Interestingly, Ras glutathiolation protects Ras from further free radical-mediated activation events. Therefore, glutathiolation does not affect Ras activity unless Ras is modified by glutathione through a radical-mediated mechanism.