The characterization of brimonidine metabolites presents some challenges since brimonidine and its metabolites generate few structurally informative fragment ions in the LC-MS/MS spectra. The objective of the current study is to use on-line hydrogen/deuterium (H/D) exchange LC-MS/MS and stable-isotope tracer techniques to further characterize unknown brimonidine metabolites in vitro and in vivo. Brimonidine and D4-brimonidine were co-incubated in rat and human microsomes and rabbit aldehyde oxidase in vitro. In addition, the urine was collected from rats co-administered orally with brimonidine and D4-brimonidine. The hepatic microsomal and urinary metabolites were then characterized by H/D LC-MS/MS system. In addition to previously characterized 2-oxobrimonidine, 3-oxobrimonidine and 2,3-dioxobrimonidine, the results show that oxidation occurs at quinoxaline ring producing oxo-hydroxybrimonidine and hydroxyquinoxaline metabolites. The hydroxyquinoxaline metabolite was only observed in microsomal incubations with hydroxylation at the 7- or 8- position. The dehydro-hydroxybrimonidine metabolites were characterized as 2-oxo or 3-oxo -4', 5'-dehydrobrimonidine. A novel metabolite ((4-bromo-lH-benzoimidazol-5-yl)-imidazolidin-2-ylidene-amine) of benzimidazole derivative of brimonidine in rats in vivo was identified and confirmed with reference standard. In conclusion, on-line H/D exchange LC-MS/MS and stable-isotope tracer techniques are useful for the characterization of brimonidine metabolites.