Buddlejasaponin IV (BS-IV), a natural triterpene saponin isolated from several herbal plants, has drawn a lot of attention for its anti-inflammatory, antinociceptive, antihyperlipidemia, and antitumor activities. In this study, a simple and sensitive method for determination of BS-IV in rat plasma was developed for the first time, using ultra-high performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS). Tenacissoside I was used as an internal standard (IS). Separation was achieved on an Agilent Extend-C18 column with gradient elution using methanol–water as mobile phase at a flow rate of 400 μL/min. A triple quadrupole mass spectrometer operating in the positive/negative ion-switching electrospray ionization mode with selection reaction monitoring (SRM) was used to determine BS-IV and IS transitions of 941.4 → 779.5 and 815.5 → 755.5, respectively. The lower limit of quantification was 3.00 ng/mL with a linear range of 3.0–3000 ng/mL. The intra- and inter-day precisions were both ≤10.4% for BS-IV, and the average intra- and inter-day accuracies ranged from −7.2% to 6.7%. The validated assay was successfully applied to a pharmacokinetic study of BS-IV following oral administration of 3, 6, 12 mg/kg and an intravenous administration of 0.9 mg/kg to rats.