Guishao Yigong Decoction (GYD), a classic prescription that tonifies the spleen and qi, has been used in clinical practices for thousands of years to treat spleen deficiency. However, its in vitro and in vivo metabolic characteristics are still unclear. In this study, a rapid and reliable analytical method based on ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS) with automated data processing (MetaboLynx) was established to investigate the metabolites and metabolic pathways of GYD in vivo and in vitro. Based on the characteristics of protonated ions, eleven precursors (ginsenoside Re, ginsenoside Rg1, ginsenoside Rb1, atractylenolide I, atractylenolide III, paeoniflorin, glycyrrhizic acid, pachymic acid, liquiritin, nobiletin, and ferulic acid) and their corresponding metabolites were detected and tentatively identified. A total of 81 metabolites were identified, including 53 metabolites in vivo, 40 metabolites of liver microsomes, and 43 metabolites of intestinal flora in vitro. Distinct metabolic patterns were observed between intestinal flora and liver microsomes - the former primarily catalyzed phase I transformations (hydroxylation, methylation, dehydroxylation, demethylation, and reduction), while the latter also executed phase II metabolism, with glucuronidation being the predominant conjugation reaction. The metabolic profiling and pathways of active components in GYD were systematically analyzed, which was helpful in clarifying its potential mechanism and clinical application.