Abstract: Postmenopausal osteoporosis (PMOP) is a prevalent age-related skeletal disorder characterized by estrogen deficiency-induced bone loss, bone microstructural deterioration, and increased fracture risk. Current therapeutic strategies such as estrogen replacement therapy are limited by potential side effects, highlighting the need for safe and effective alternatives derived from traditional Chinese medicine (TCM). The Cuscuta chinensis-Lycium barbarum herb pair, a core combination in TCM formulas for nourishing the kidney and replenishing essence, has demonstrated anti-PMOP activity and phytoestrogenic effects in previous studies. However, its in vivo pharmacodynamic active constituents and metabolic mechanisms under pathological states remain unclear, which hinders its clinical application and drug development. This study aimed to systematically identify the prototype components and metabolites of the herb pair in PMOP model rats and clarify their hepatic metabolic pathways using ultra-performance liquid chromatography tandem quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF MS). Firstly, PMOP models were established by bilateral ovariectomy, and the active fraction of the herb pair was extracted with 50% ethanol. After intragastric administration (4.2 g/kg, for 5 consecutive days), samples of hepatic portal vein blood, bile, and abdominal aorta blood were collected and processed for subsequent analysis. Subsequently, a method of UPLC-Q-TOF MS was developed using acetonitrile (A) and 0.1% formic acid aqueous solution (B) as mobile phases at a flow rate of 0.3 mL/min, with gradient elution. Mass spectrometry was equipped with an electrospray ionization (ESI) source and operated in both positive and negative ion modes over a mass range of m/z 50-1 200. Finally, the acquired data were analyzed using Waters MassLynx V4.1, and chemical components were identified based on quasi-molecular ions, fragment ions, fragmentation pathways, database records, and reference standard information. A total of 93 components were identified in the three biological samples, including 39 prototype components and 54 metabolites. Specifically, 76 components (39 prototypes and 37 metabolites) were detected in the hepatic portal vein, 78 components (37 prototypes and 41 metabolites) were detected in bile, and 67 components (35 prototypes and 32 metabolites) were detected in the abdominal aorta. The main metabolic pathways included Phase I metabolism (e.g., hydroxylation, dehydroxylation, hydrolysis), Phase II metabolism (e.g., glucuronidation, methylation, sulfation), and their combined biotransformation reactions. The anti-PMOP active fraction of the Cuscuta chinensis-Lycium barbarum herb pair underwent a series of metabolic reactions in rat liver tissue, generating abundant metabolites. This study elucidated the dynamic biotransformation process of the pharmacodynamic substances of the Cuscuta chinensis-Lycium barbarum herb pair (for anti-PMOP effects) in the liver, providing experimental evidence to reveal its material basis of efficacy.