S-nitrosylation is the binding of an NO group to a cysteine or other thiol. Like phosphorylation, S-nitrosylation is a precisely targeted and rapidly reversible post-translational modification that serves as an on/off switch for protein function during cell signaling. However, unlike phosphorylation, S-nitrosylation of proteins occurs nonenzymatically and is mediated, at least in part, by redox-regulated chemical reactions in cells. Alterations in pH, pO(2), cellular reductants, transition metals, and UV light lead to the loss and/or gain of S-NO bonds. Due to the redox-sensitive nature of the modification, analysis of protein S-nitrosylation is technically difficult, since the S-NO bond is easily disrupted during sample preparation. In addition, the level of S-nitrosylated proteins in cells approaches the limit of detection of currently available technology. Despite these technical challenges, several useful methods have been developed recently to measure protein S-nitrosylation in biological samples, and these are described in this unit.