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S-nitrosothiols (RSNOs) are very important biomolecules that play crucial roles in many physiological and physiopathological processes. They act as NO-donors and are candidates for future medicines. Their identification and quantitation are therefore important for biomedical applications. One, two or more RSNOs can then be combined to design a drug and therefore, the quantification of each is important to establish an acceptable quality control process. Till date, miniaturized devices have been used to detect RSNOs based on their total quantitation without a preceding separation step. This study reports on an original and integrated microdevice allowing for the successive electrokinetic separation of low molecular weight RSNOs, their decomposition under metal catalysis, and their quantitation by amperometric detection of the produced nitrite in the end-channel arrangement, leading to their quantitation in a single run. For this purpose, a commercial SU-8/Pyrex microfluidic system was coupled to a portable and wireless potentiostat. Different operating and running parameters were optimized to achieve the best analytical data, allowing for an LOD equal to 20 μM. The simultaneous separation of S-nitrosoglutathione and S-nitrosocysteine was successfully obtained within 75 s. The proposed methodology using SU-8/Pyrex microfluidic devices opens new possibilities to investigate future drug candidates for NO-donors. [ABSTRACT FROM AUTHOR]