Background: Yiganmingmu oral liquid (YGMM), a well known over-the-counter (OTC) drug in China, is composed of 12 types of valuable herbal medicines and has been widely used in clinical for the treatment of soreness and weakness of waist and knees, dizziness, memory loss, and fatigue. However, the chemical compositions of YGMM and its absorbed compounds in plasma remain unclear. Methods: Since chemical investigation is the first important step to reveal effects and action mechanisms of traditional Chinese medicine (TCM), in this study, based on the self built components database, systematic characterization of the chemical profile of YGMM in vitro was carried out by using a reliable UPLC-Q-TOF-MS method. Moreover, to obtain better understanding of the absorbed prototypes in plasma, serum pharmacochemistry analysis of YGMM after oral administration was conducted by using cynomolgus monkeys as animal model. Results: A total of 667 constituents from the 12 single herbal medicines were collected in the self built components database by searching the reported literatures, and 415 of them were initially screened as candidate compounds in YGMM by comparison of their experimental accurate mass measurements with those theoretical values. After that, 117 compounds including 17 phenolic acids, 25 flavonoids, 4 alkaloids, 10 phthalides, 5 monoterpenes, 8 triterpenoid saponins, 9 anthraquinones, and 39 other compounds, were unambiguously identified or tentatively characterized by analysing their MS/MS fragmentation patterns, and also by comparison with reference standards and those data reported in the literatures. 61 prototypes absorbed in plasma of cynomolgus monkey, including 13 phenolic acids, 21 flavonoids, 8 phthalides, 3 monoterpenes, 4 triterpenoid saponins, and 12 other compounds were tentatively assigned by serum pharmacochemistry analysis after oral administration. Conclusion: It was the first comprehensive analysis of chemical constituents of YGMM and prototypes in plasma, and the data analysis strategy developed in this study showed high efficiency in the structural elucidations. The results might provide scientific evidence for further research on material basis of YGMM. © 2021, The Author(s).