Acrylamide (AA) is reported present in high-temperature-processed food and classified as a possible human carcinogen. In vivo metabolic activation of AA by CYP 2E1 to glycidamide (GA) may play an important role on AA carcinogenicity. AA and GA can be detoxified by glutathione-S-transferase to form AA and isomeric GA glutathione conjugates (AA-, GA2- and GA3-GSH, respectively), which can be further metabolized to mercapturic acids (MAs). Although many studies analyzed MAs in urine of rodents and humans, few studies have characterized and analyzed the GSH conjugates. The objectives of this study were to synthesize, purify, and characterize AA-GSH, GA2-GSH, GA3-GSH, ((13)C3)-AA-GSH, ((13)C3)-GA2-GSH, and ((13)C3)-GA3-GSH to develop an isotope-dilution liquid chromatography tandem mass spectrometry (LC-MS/MS) method to analyze AA- and GA-GSHs in blood of rats treated with AA. After purification and characterization of these conjugates, the LC-MS/MS method was developed and validated. This method reveals a limit of detection (S/N=3) at 0.017 and a limit of quantitation (S/N=10) at 0.05ng/mL of serum for AA-GSH, 0.075 and 0.25ng/mL for GA2-GSH, and 0.15 and 0.5ng/mL for GA3-GSH. Analyzed with this method, AA-GSH, GA2-GSH and GA3-GSH were 1651.1±374.5, 18.4±6.3 and 75.3±31.3ng/mL in blood of male rats at 2h after treatment with 5mg/kgbw of AA by ip injection. These results showed that the LC-MS/MS method was successfully developed to analyze AA-GSH, GA2-GSH and GA3-GSH with satisfying sensitivity of AA and GA which were conjugated by glutathione in vivo.