2α, 3α, 24-Trihydroxyurs-12-en-28-oic acid (TEOA) is a prominent ursane-type triterpenoid isolated from the root of Actinidia deliciosa, which has numerous pharmacological activities. To date, there is no report on the pharmacokinetic characterization of TEOA in biological samples. A specific, sensitive and robust liquid chromatography-tandem mass spectrometry (LC-MS-MS) method is reported for the fast assay of TEOA in rat plasma. Plasma sample preparation was based on liquid-liquid extraction with ethyl acetate. The extracted samples were determined using LC-MS-MS coupled with an atmospheric-pressure chemical ionization source in multiple-reaction monitoring scan mode. Chromatographic separation was achieved on a C18 Capcell PAK UG120 column (100 × 4.6 mm, i.d., 5 µm) using an isocratic elution mode with a total running time of 2.8 min. The selected ion pairs m/z 471.3 → 203.1 for the analyte and m/z 309.2 → 163.1 for internal standard were monitored in positive ionization mode for MS-MS detection. This method has been fully validated and the results showed good linearity, precision, accuracy and recovery. A stability test was conducted under various sample preparation, analysis and storage conditions. The method was successfully applied for the pharmacokinetic study of TEOA in rat plasma after oral administration of 100 mg/kg TEOA.