Abstract: Identification of metabolites of Danshen-Honghua herb pairs in isolated rat intestinal flora based on HPLC-Q-TOF-MS/MS technique. By incubating enterobacteria in isolated rats as well as inactivated enterobacteria in the incubation solution. The extracts of Danshen-Honghua herb pairs were added separately and co-incubated under anaerobic conditions. Animal experiments and protocols were approved by the Laboratory Animal Ethics Committee of Shaanxi University of Traditional Chinese Medicine (approval number: TCM-2020-030-E05). A total of 14 compounds, including 5 prototypes and 9 metabolites, were identified in the isolated rat intestinal incubation fluid. In contrast, no metabolites were detected in the inactivated enterobacterial fluid, except for the prototype component. The results showed that the main components of the Danshensu, salvianolic acid B, rosmarinic acid, lithospermic acid, and hydroxysafflor yellow A, could be metabolized by the intestinal flora, and these active ingredients were mainly metabolized in the rat intestinal flora in isolation by hydroxylation, decarboxylation, deoxygenation, decarboxylation and dehydration in phase Ⅰ, sulfate esterification and methylation in phase Ⅱ. This proved that the Danshen-Honghua herb pair could be transformed into various metabolites by the action of rat intestinal flora, further clarifying the role of intestinal flora in the metabolic transformation of the active ingredients of Chinese medicine and laying the foundation for perfecting the potent substances of the pair.