S-nitrosylation (SNO) is an important oxidative posttranslational modification in the regulation of cardiac mitochondria. SNO modification of several mitochondrial proteins has been associated with cardiac preconditioning and improved cell survival following ischemia/reperfusion injury. Due to their labile nature, SNO modifications are challenging to study using traditional biochemical techniques; particularly, the identification of individual modified cysteine residues. Here, we describe the details of the cysTMT 6 switch assay, a variation of the classic biotin switch protocol. The cysTMT 6 reagent provides a simplified and powerful approach to SNO detection by combining unambiguous identification of the modified cysteine residue and relative quantification of up to six samples by mass spectrometry analysis.