Abstract: OBJECTIVE To analyze and identify the metabolites of isoimperatorin in normal rats in vivo. METHODS After the rats were intragastrically administered, the plasma, urine and feces samples of rats in normal and dosed groups were collected, and the samples were processed by solid phase extraction. Then, UHPLC-HRMS(Q-Exactive Orbitrap MS) high- resolution mass spectrometry was used to analyze biological samples, and the metabolites were screened and identified. RESULTS In both positive and negative ion modes, a total of 32 metabolites were accurately analyzed and identified based on the precise molecular masses obtained, chromatographic retention behavior, control mass spectral cleavage patterns, characteristic fragment ions, and relevant literature reports. The main metabolic pathways were furan ring opening, lactone hydrolysis, oxidation, methylation, glucuronidation, sulfation and their complex reactions. Among them, the glucuronidation product was discovered for the first time. CONCLUSION This study comprehensively clarifies the transformation pathways of isoimperatorin in rats, which can provide a basis for its pharmacodynamic study and metabolism study analysis of the other furocoumarins.