Identifying the compound formulae-related xenobiotics in bio-samples is full of challenges. Conventional strategies always exhibit the insufficiencies in overall coverage, analytical efficiency, and degree of automation, and the results highly rely on the personal knowledge and experience. The goal of this work was to establish a software-aided approach, by integrating ultra-high performance liquid chromatography/ion-mobility quadrupole time-of-flight mass spectrometry and in-house high-definition MS2 library, to enhance the identification of prototypes and metabolites of the compound formulae in vivo, taking Sishen formula (SSF) as a template. Seven different MS2 acquisition methods were compared, which demonstrated the potency of a hybrid scan approach (namely HDDIDDA) in the identification precision, MS1 coverage, and MS2 spectra quality. The HDDIDDA data for 55 reference compounds, four component drugs, and SSF, together with the rat bio-samples (e.g., plasma, urine, feces, liver, and kidney), were acquired. Based on the UNIFI™ platform (Waters), the efficient data processing workflows were established by combining mass defect filtering (MDF)-induced classification, diagnostic product ions (DPIs), and neutral loss filtering (NLF)-dominated structural confirmation. The high-definition MS2 spectral libraries, dubbed in vitro-SSF and in vivo-SSF, were elaborated, enabling the efficient and automatic identification of SSF-associated xenobiotics in diverse rat bio-samples. Consequently, 118 prototypes and 206 metabolites of SSF were identified, with the identification rate reaching 80.51% and 79.61%, respectively. The metabolic pathways mainly involved the oxidation, reduction, hydrolysis, sulfation, methylation, demethylation, acetylation, glucuronidation, and the combined reactions. Conclusively, the proposed strategy can drive the identification of compound formulae-related xenobiotics in vivo in an intelligent manner.