AbstractIntroduction – Xuebijing injection (XBJ) is a traditional Chinese herbal prescription widely used in the treatment of sepsis. This is the first report concerning the identification of XBJ constituents. In addition, to evaluate XBJ's quality, partial least square discrimination analysis (PLS‐DA) was performed on chemical fingerprint data.Objective – Establish an LC‐MS method to identify the components in XBJ for the purpose of quality control.Methodology – Compounds were separated by HPLC using a C18 column and gradient elution of acetonitrile–methanol (60:40, v/v) and water–acetic acid (100 : 0.5, v/v) in 80 min. HPLC equipped with diode array detector (DAD) coupled with time‐of‐flight (TOF) tandem mass spectrometry and HPLC electrospray ionisation (ESI) multi‐stage tandem ion‐trap mass spectrometry (IT‐MSn) method was developed to analyse XBJ's major components. Both positive and negative ionisation modes were employed.Results – Twenty‐one compounds including amino acids, phenolic acids, flavonoid glycoside, terpene glycoside and phthalide were identified or tentatively characterised. Their retention times, UV and MS spectra were compared with those of authentic compounds or literature data. The score plot of PLS‐DA clearly revealed variations among samples produced in different commercial batches.Conclusion – The analytical method developed is highly effective for the discrimination and quality control of XBJ.