Background: Tripterygium wilfordii (TW) is widely employed to treat rheumatoid arthritis and autoimmune disorders clinically, which, however, accompany with disturbing hepatotoxicity and nephrotoxicity. The previous research showed that Panax notoginseng (PN) compatibly and significantly reduces the TW-induced hepatotoxicity. Objective: To explore the underlying mechanism, the present study was designed to reveal the influence of PN on the intestinal absorption process of TW-derived active components in rat. Materials and Methods: An in situ single-pass intestinal perfusion technique was established and preformed to obtain the perfusate samples of triptolide (TP), tripterine (TE), TW extract, and TW-PN extract. A rapid and sensitive ultra-performance liquid-chromatography tandem mass spectrometry method was subsequently developed and validated to determine the concentrations of TP and TE in the perfusate samples. Then, the absorption parameters, effective permeability, absorption rate constant, and percentage of 10 cm intestinal absorption were calculated strictly. Results: The final data indicated that both TP and TE have no special absorption site in the intestine and are primarily absorbed in a passive manner. Otherwise, the absorption of TP was decreased from compatibility of PN, but the absorption of TE was enhanced. Conclusion: The absorption reduction of TP and absorption elevation of TE from TW initiated by the combination of PN are contributed to attenuate the toxicity and reinforce the therapeutic efficacy of TW. It is practically reasonable of usage of TW compatibility with PN clinically.