A dynamic microdialysis sampling method with liquid chromatography–quadrupole time-of-flight mass spectrometry (Q-TOF-MS) was developed for rapid and sensitive analysis of the metabolite profile of Panax notoginseng extract (PNE) in rat bile. In vivo studies in male Sprague-Dawley rats were performed with microdialysis probes implanted into the bile duct before bile samples were collected from 0 to 12h. Metabolites of PNE were identified using dynamic adjustment of the fragmentor voltage to produce structure-relevant fragment ions. The mass accuracy of precursor and fragment ions was typically within 5ppm of the theoretical values. We identified 7 compounds: 4 parent compounds (notoginsenoside R1, ginsenosides Rg1, Rb1, and Rd) and 3 metabolites (ginsenosides Rg2, Rh2, and compound K). Data from this study suggest that this microdialysis technique could be used in notoginseng saponin metabolic animal studies.