The present study examined the impact of the supplementation of glutathione (GSH), γ-L-glutamyl-L-cysteinyl-glycine, on human blood GSH levels. Healthy human volunteers were orally supplemented with GSH (50 mg/kg body weight). Venous blood was collected from the cubital vein before and after ingestion. Plasma was mixed with 3 volumes of ethanol. The supernatant and precipitate were used for the deproteinized and protein fractions of plasma, respectively. Blood cell and plasma fractions were pretreated with 5% trichloroacetic acid-2% 2-mercaptoethanol to reduce the oxidized form of GSH and liberate protein-bound GSH. The 2-mercaptoethanol-pretreated GSH was determined by precolumn derivatization with 6-aminoquinolyl-N-hydroxy succinimidyl carbamate and liquid chromatography-tandem mass spectrometry. There was no significant difference in GSH contents in the deproteinized fraction of plasma and blood cell fraction after GSH ingestion. However, the GSH contents in the protein-bound fraction of plasma significantly (P<0.01) increased from 60 to 120 min after GSH supplementation.