Oxidative stress plays a role in the development of chronic peripheral arterial disease (PAD) because under these conditions redox regulation is impaired, inducing the S-glutathionylation of proteins. A method of estimating the levels of S-glutathionylated proteins has been developed using biotinylated glutathione S-transferase, which allows the study of their crucial role in the oxidative stress-related progression of PAD.The serum levels of S-glutathionylated proteins were examined in 41 patients with arteriosclerosis obliterans (ASO) and 38 age-matched non-ASO patients using biotinylated glutathione S-transferase. The levels were higher in the patients with ASO, even early on, and positively correlated with the ankle/brachial index. In vitro, the levels of S-glutathionylated proteins were reduced in the presence of glutathione and glutaredoxin.Serum levels of S-glutathionylated proteins are a sensitive risk-marker for ASO at an early stage.