Abstract: OBJECTIVE To establish methods for the determination of salvianolic acid B metabolites in rats' plasma, bile, urine and feces, and investigate their metabolic pathways. METHODS Eighteen SD rats were equally divided into two groups:intragastric administration and intravenous injection. Each group was divided into three groups to collect plasma, bile, urine and feces, respectively. Two rats of each group were given salvianolic acid B single-dose by intragastric administration(500 mg·kg^-1) and tail vein injection(16.5 mg·kg^-1) respectively, and the other one was administered gavage water and saline intravenously as a blank control. Determine the metabolites of salvianolic acid B in plasma, bile, urine and feces by HPLC-Q-TOF-MS/MS, and infer the metabolic pathways. RESULTS According to information of MS and MS², eight metabolites of salvianolic acid B were found in rats, including eight metabolites in bile and feces, three metabolites in urine and two metabolites in plasma. Based on the structure of salvianolic acid B and its metabolites, the in vivo metabolism of salvianolic acid B was mainly metabolized by methylation reaction, ester bond hydrolysis reaction. CONCLUSION The established analytical method is accurate, sensitive and rapid, and meets the requirements of biological sample analysis. It is applicable to the analysis of the metabolites of salvianolic acid B in rat plasma, bile, urine and feces, and 8 metabolites were identified. Metabolic experiments show that bile excretion is the most important excretion of salvianolic acid B.