Tripterygium wilfordii, which contains a lot of biological toxic compounds such as Wilfordine and Wilforine, is one of the toxic nectar plants. The Wilfordine and Wilforine may be transferred to honey by honey bees. Due to the low content and complex matrix, determination of Wilfordine and Wilforine in honey is not easy. In this study, a highly sensitive method based on liquid-liquid extraction (LLE) and LC-MS/MS has been developed. The results showed that the detection limits of Wilfordine and Wilforine in honey sample were 5.16 and 10.80 ng/kg, respectively.