To study a vitro extraction method to determine soluble mercury and mercury species in Angong Niuhuang Wan and investigate a vitro safety evaluation method, the best extraction scheme was determined after a great deal of conditioning experiments focusing on how the solvent, purification, duration of extraction and purification etc impact on mercury extraction. Soluble mercury was determined by inductively coupled plasma mass spectrometry and mercury species were determined by high performance liquid chromatography/inductively coupled plasma mass spectrometry. Result show that the best extraction method of soluble mercury and its species was ultrasonic extraction in artificial intestinal juice at 37 ℃ for 2 hours, letting it stand for 20 hours and taking the supernatant. The method was reproducible, which could simulate the human body environment to maximum extent. Methyl mercury, ethyl mercury and Hg2+ were not found in the extracts of Angong Niuhuang, although the content of soluble mercury was high. It was concluded that the method could be applied to the vitro extraction and determination of soluble mercury and mercury species in Chinese traditional medicines containing cinnabar. Methyl mercury, ethyl mercury and Hg2+ could be taken as the targets of in vitro safety evaluation of Angong Niuhuang.