Abstract: A rapid analytical method for the simultaneous determination of triptolide, wilforgine and wilforine was developed using high performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS). The contents of those three active substances in different tissues and plant tissue culture of Tripterygium wilfordii were compared. The ultrasonic extraction and reflux extraction technologies were used for extraction of those three active substances in T. wilfordii. In the reflux extraction process, the analytes were extracted by V (methanol) : V (acetonitrile) = 1:1 solution, and cleaned up by Supelclean LC-Si solid phase extraction cartridges. During the ultrasonic extraction, the analytes were extracted by ethanol, purified by OASIS HLB SPE column, and separated on a reversed phase C₁₈ column with gradient elution with acetonitrile and water as the mobile phase. The extract was then determined with HPLC-ESI-MS/MS. The results showed that the highest contents of triptolide is detected in adventitious roots, followed by hairy root and root bark, while very low content was detected in stems and leaves. The highest contents of wilforgine content is observed in hairy root, followed by the root bark, and it was not detected in leaves. Wilforine contents determined in hairy root and root bark are similar. When the addition level ranged from 0.01 mg/kg to 2 mg/kg, the recoveries of three active substances of T. wilfordii was 81%-109%, and with RSD ranged from 0.4% to 1.8% (n = 5). The detection limits of the three active substances ranged from 0.08 μg/mL to 0.12 μg/mL. Results indicated that this method is highly efficient, with good sensitivity and accuracy, which can quickly monitor the quality of T. wilfordii extract and its products.