Several biomarkers of oxidative stress have been proposed and used in clinical research but so far unreliable or, at least, controversial results have been obtained. Given the high susceptibility of sulfhydryl groups to oxidation, we here suggest the use of a protein thiolation index (PTI), i.e., the molar ratio between the sum of all low molecular mass thiols bound to plasma proteins (forming, as a whole, S-thiolated proteins) and protein free cysteinyl residues, as a suitable biomarker of oxidative stress. While titration of free thiols can be performed by a simple spectrophotometric procedure, accurate quantification of S-thiolated proteins is problematic and current methods require, in most cases, application of time-consuming chromatographic techniques, making their application to large-scale clinical studies difficult. Here we report a new spectrophotometric method which relies on the specific determination of low molecular mass thiols released from S-thiolated proteins after dithiothreitol reduction. These amino acids can be titrated by conjugation with ninhydrin which, reacting with primary and secondary amine groups, yields a deep blue-purple color, which can be spectrophotometrically revealed. PTI showed an age dependency with a near linear increase during aging in humans. In addition, PTI was significantly higher in patients suffering from alkaptonuria with respect to healthy controls, suggesting that increased prooxidant conditions occur in the blood of these subjects.