S-Nitrosothiols (RSNOs) are carriers of nitric oxide (NO) and have important biological activities. We propose here the use of gold nanoparticles (AuNPs) and NO-selective amperometric microsensor for the detection and quantification Of S-nitro-soglutathione (GSNO) as a step toward the determination of plasma RSNOs. AuNPs were used to decompose RSNOs with the quantitative release of free NO which was selectively detected, with a NO microsensor. The optimal [GSNO]/[AuNPs] ratio was determined, corresponding to an excess of AuNP surface relative to the molar GSNO amount. Moreover, the influence of free plasma thiols on this method was investigated and a protocol based on the blocking of free thiols, with iodoacetic acid, forming the carboxymethyl derivative of the cysteine residues, is proposed.