Tongmai Sini decoction (TSD), the classical prescriptions of traditional Chinese medicine, consisting of three commonly used herbal medicines, has been widely applied for the treatment of myocardial infarction and heart failure. However, the absorbed components and their metabolism in vivo of TSD still remain unknown. In this study, a reliable and effective method using ultra-performance liquid chromatography coupled with hybrid quadrupole-Orbitrap mass spectrometry (UHPLC-Q-Exactive-MS/MS) was employed to identify prototype components and metabolites in vivo (rat plasma and urine). Combined with mass defect filtering (MDF), dynamic background subtraction (DBS), and neutral loss filtering (NLF) data-mining tools, a total of thirty-two major compounds were selected and investigated for their metabolism in vivo. As a result, a total of 82 prototype compounds were identified or tentatively characterized in vivo, including 41 alkaloids, 35 phenolic compounds, 6 saponins. Meanwhile, A total of 65 metabolites (40 alkaloids and 25 phenolic compounds) were tentatively identified. The metabolic reactions were mainly hydrogenation, demethylation, hydroxylation, hydration, methylation, deoxylation, and sulfation. These findings will be beneficial for an in-depth understanding of the pharmacological mechanism and pharmacodynamic substance basis of TSD.