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Euonine and wilforidine are biologically active Tripterygium wilfordii Hook. f. alkaloids. In this paper, a rapid and sensitive high-performance liquid chromatographymass spectrometry (MS) method was developed and validated for the simultaneous determination of trace euonine and wilforidine in human plasma. An Oasis mixed-mode cation-exchange polymeric sorbent was used for solid-phase extraction. The separation was carried out on a reversed-phase Zorbax Plus C-18 column (50 mm 2.1 mm, 1.8 m) by using ammonium acetate (5 mmol/L)/acetonitrile (30/70, v/v) as the mobile phase at a flow rate of 0.7 mL/min. The quantification was carried out via ion trap MS in the positive selected ion monitoring mode using aconitine as an internal standard. Calibration curves showed good linearity ranging from 0.5 to 100.0 g/L with correlation coefficient values 0.9990. The average recoveries of euonine and wilforidine ranged from 85.4 to 101.0 and 92.0 to 97.5, respectively. The intra- and interday relative standard deviations were 8.7 and 12.9, respectively. The limits of quantification for both euonine and wilforidine were 0.5 g/L, which is suitable in the clinical pharmaceutical research of volunteer patients with rheumatoid arthritis.