Abstract: The hepatic sequential metabolism research model was employed to delineate the distribution profiles and hepatic metabolism patterns of the estrogen-like active fractions from Cuscuta chinensis. This study specifically aims to compare the distribution and metabolism features of the active fractions before and after they enter the liver under estrogen deficiency conditions. An estrogen deficiency model was first established in female rats via bilateral ovariectomy. Following this, intragastric administration of the tested fractions was performed, and various biological samples were systematically collected for subsequent analysis. The metabolic profiles of Cuscuta chinensis effective fractions in the sequential system of “hepatic portal vein blood-hepatic homogenate-bile-abdominal aorta blood” were constructed by ultra high performance liquid chromatography-quadrupole-time of flight mass spectrometry (UPLC-Q-TOF MS). Based on the data acquired under positive and negative ion modes, as well as comparison with the mass spectral information of the control group, the UNIFI database and a self-constructed database, the prototype components and metabolites were characterized in accordance with the compounds’ fragmentation patterns, and their metabolic pathways and distribution characteristics were further analyzed. Comprehensive profiling of the Cuscuta chinensis active fractions enabled the identification of 75 chemical components. These included flavonoids, organic acids, phenylpropanoids, alkaloids, and sugars, together with other compounds belonging to additional structural classes. In the estrogen-deficient rat model, a total of 44 prototypical components and 66 metabolites were successfully characterized. Most of the identified compounds belonged to the flavonoid class. The identified metabolic pathways involved both Phase I and Phase II reactions, including but not limited to dehydroxylation, hydroxylation, glucuronidation, and glycine conjugation. Notably, glucuronidation was identified as the core metabolic pathway that mainly mediated the transformation of flavonoid constituents. Hepatic sequential metabolic analysis demonstrated that a variety of prototype components were capable of crossing the intestinal mucosal barrier, thereby reaching the liver and entering the systemic circulation. This finding indicated their potential to exert systemic pharmacological effects in vivo. Moreover, this study revealed that a subset of prototype components underwent rapid hepatic uptake and sequestration during the initial stage. Consequently, these components were no longer involved in further biotransformation processes. Based on the metabolic data, it is also postulated that both kaempferol and luteoloside may undergo enterohepatic circulation. The present investigation has systematically delineated the characteristic hepatic metabolic behaviors and pathways of the Cuscuta chinensis active fractions under estrogen-deficient conditions, providing key scientific insights into clarifying their entire in vivo biotransformation process and the material basis of its pharmacological activity.