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A rapid and simple background-free high-performance liquid chromatography/ electrospray ionization tandem mass spectrometry (HPLC/ESI-MS/MS) approach has been developed for simultaneously determining free thiol compounds including glutathione (GSH), cysteine (Cys), N-acetylcysteine (NAC), homocysteine (Hcy), and cysteinylglycine (CysGly) in biological samples.