The direct analysis of cyanide (HCN or CN- inclusively symbolized as CN) to confirm exposure has major limitations due to cyanide's volatility, reactivity, and short half-life in biological fluids. These limitations have led to the exploration of cyanide detoxification products for indirect verification of cyanide exposure. Although cyanide interacts strongly with sulfur-containing molecules, to date, biomarkers resulting from the interaction of cyanide with glutathione (GSH; i.e., a biologically abundant sulfur-donating biomolecule) have yet to be discovered. In this study, we studied the interaction of CN and GSH to produce 2-aminothiazoline-4-oxoaminoethanioc acid (ATOEA). An LC-MS/MS method was developed and validated to analyze ATOEA from plasma, producing a linear range of 0.5-50 μM, a limit of detection of 200 nM, and excellent precision and accuracy. ATOEA concentrations were significantly elevated in the plasma of animals following cyanide exposure. Moreover, the production of ATOEA from cyanide exposure was confirmed by detection of both ATOEA and ATOEA-13C15N in rabbit plasma ( N = 11 animals) following administration of NaCN:K13C15N (1:1), with a similar amount of ATOEA and ATOEA-13C15N formed ( R2 = 0.9924, p < 0.05). The concentration of ATOEA increased with cyanide dose and then decreased rapidly when an antidote was administrated. This study definitively showed that ATOEA is produced from interaction of CN and GSH and can serve as a biomarker of cyanide exposure.