A simple, sensitive and rapid method using high performance liquid chromatography-tandem mass spectrometry (HPLC–MS/MS) was developed and validated for quantification of glycine-β-muricholic acid (Gly-MCA) in mouse plasma for the first time. Plasma samples were pretreated with protein precipitation. The analyte and internal standard were separated on a Shimadzu Shim-pack XR-ODS column (4.6 × 50 mm, 2.2 μm) using 0.1% formic acid-water-methanol as mobile phase, with a runtime of 5 min. Detection was performed using negative ion electrospray tandem mass spectrometry via multiple reaction monitoring (MRM) scan mode. The linear range was 5 ng–2 μg/ml (correlation coefficient >0.995) for Gly-MCA with a lower limit of quantitation of 5 ng/ml. The intra-day and inter-day precision were less than 9.2% for the analyte and accuracy was from 0.4% to 7.0%. This analytical method was then successfully applied to a pharmacokinetic study of Gly-MCA following oral administration and intraperitoneal injection in mice.