Protein thiol modifications occur under both physiological and pathological conditions and have been shown to contribute to changes in protein structure, function, and redox signaling. The majority of protein thiol modifications occur on cysteine residues that have a low pKa; these nucleophilic proteins comprise the "reactive thiol proteome." The most reactive members of this proteome are typically low-abundance proteins. Therefore, sensitive and quantitative methods are needed to detect and measure thiol modifications in biological samples. To accomplish this, we have standardized the usage of biotinylated and fluorophore-labeled alkylating agents, such as biotinylated iodoacetamide (IAM) and N-ethylmaleimide (NEM) and BODIPY-labeled IAM and NEM, for use in one- and two-dimensional proteomic strategies. Purified fractions of cytochrome c and glyceraldehyde-3-phosphate dehydrogenase were conjugated to a known amount of biotin or BODIPY fluorophore to create an external standard that can be run on standard SDS-PAGE gels, which allows for the quantification of protein thiols from biological samples by Western blotting or fluorescence imaging. A detailed protocol is provided for using thiol-reactive probes and making external standards for visualizing and measuring protein thiol modifications in biological samples. © 2009 Elsevier Inc. All rights reserved.