A simple and sensitive method to determine thiosulfate in human blood and urine was devised. Thiosulfate was first alkylated with pentafluorobenzyl bromide, with L-ascorbic acid as the stabilizer and sodium chloride as the catalyst. The alkylated thiosulfate was oxidized with iodine to obtain bis(pentafluorobenzyl)disulfide, which was then subjected to gas chromatography and gas chromatography/mass spectrometry. Mass fragmentography was used to identify the disulfide, and gas chromatography with an electron capture detector was used for quantitative determination. The lower limit of detection was 0.003 mumol/mL.