An improved bile microdialysis sampling technique was established and coupled with liquid chromatography quadrupole time-of-flight mass spectrometry (LC-qTOF-MS) analysis. This method was successfully applied to investigate the metabolic profiles of Ermiao wan (EMW) formula in the bile of Sprague-Dawley (SD) rats. Based on accurate mass information and fragment patterns, 23 alkaloids and lactones metabolites were tentatively identified. Their metabolic pathway involved in glucuronidation, sulfation, hydroxylation and hydrolysis. Because of the high time resolution of microdialysis, the metabolic profiles of EMW were also investigated. Jatrorrhizine, columbamine and other components showed a "double-peak" profiles, suggesting the existence of enterohepatic circulation. The developed microdialysis sampling/ LC-qTOF-MS method provides a simple and efficient research tool for understanding and clarifying the mechanism of hepatobiliary excretion of complex components. CI - Copyright (c) 2020 Elsevier B.V. All rights reserved.