To study the metabolites of glycyrrhetic acid (GA) in rat bile, an analytical method was developed to identify GA and its metabolites by liquid chromatography mass spectrometry (LC-MS). Rat bile was collected after i.v. injection of GA. Three major GA-related peaks were detectable in rat bile by high-performance liquid chromatography (HPLC) analysis at 254 nm. LC-MS spectra showed their protonated molecular ions at m/z 727, 647, and 551. Furthermore, the three metabolites were also confirmed to exist in rat bile on LC-MS total ion chromatogram (TIC). Taken together with the susceptible nature to beta-glucuronidase digestion and alkaline conditions, they were identified to be a novel sulfate-glucuronide diconjugate and the known monoglucuronide and sulfate conjugate, respectively.