Xuebijing injection (XBJ) is a traditional Chinese herbal prescription widely used in the treatment of sepsis. Extensive studies revealed that the major bioactive constituents of XBJ injection, including phenolic acids, flavonoids, monoterpene glycosides, lactones and organic acids, play an important role in the treatment. In this study, a rapid, sensitive and accurate ultra high performance liquid chromatography – Q Exactive hybrid quadrupole – orbitrap high-resolution accurate mass spectrometry (UHPLC-Q-Orbitrap HRMS) method was developed for simultaneous determination of twelve bioactive compounds in rat plasma after intravenous administration of XBJ injection. A gradient elution for separation was achieved on a waters ACQUITY UHPLC® BEH C18 column (2.1 mm × 50 mm, 1.7 μm) column with acetonitrile-water (containing 0.1% formic acid) as mobile phase at a flow rate of 0.2 mL/min. All compounds and IS were monitored by parallel reaction monitoring assay both in positive and negative ion mode. The developed method was validated for intra-day and inter-day accuracy and precision whose values fell in the acceptable limits. Extraction recoveries at three levels of QC concentrations were all more than 80% for all compounds and IS, and matrix effects were found in the range of 80.0–120.0%. Stability results showed that all analytes were stable at the investigated conditions. The validated method was successfully applied to a pharmacokinetic study of Xuebijing injection following intravenous administration of 2.5, 5.0, 10.0 mL/kg to rats respectively. And the result indicated that the pharmacokinetic behavior of XBJ injection is positively related to dosage at the range of 2.5–10 mg/kg. This study will provide a meaningful basis for evaluating the rationality of XBJ injection for clinical application.