Abstract: OBJECTIVE To investigate the in vitro stability of timosaponin BⅡ in different physiological pH and biological sample.METHODS Timosaponin BⅡ was incubated in different phosphate buffer solutions with pH of 1.2, 6.8, 7.4 and 8.0, artificial gastrointestinal fluid, artificial gastrointestinal with no pepsin and trypsin and isolate biological media. The concentration of timosaponin B was determined Ⅱ by HPLC in order to investigate the degradation and the enzymatic stability of timosaponin B. ⅡRESULTS Timosaponin BⅡ was stable in pH 1.2, 6.8, 7.4 and 8.0, artificial gastrointestinal fluid, artificial gastrointestinal with no pepsin and trypsin within 9 h. Obvious degradation of timosaponin BⅡ was observed after incubationed in the large intestine and small intestine and their contents of isolated rats.CONCLUSION Timosaponin B is stable in Ⅱ simulated conditions of human stomach, intestines fluids, with no degradation and influence by pepsin and trypsin. But obvious degradation of timosaponin BⅡ is observed after incubationed in the large intestine and small intestine and their contents of isolated rats. Unaffected by pepsin and trypsin, it is affected by intestinal flora.