Abstract: The objective of this study to explore the active components and action mechanisms of Tufuling Qiwei powder (TFLQW) in the treatment of ulcerative colitis (UC) based on serum migration components, network pharmacology and experimental validation. The UC mouse model was induced using dextran sulfate sodium (DSS), and TFLQW was administered at different concentrations via gavage. The inflammatory factors in mouse serum were detected using the ELISA, and the mouse colon tissues were evaluated with HE staining and pathological tissue scoring to confirm that TFLQW could alleviate the UC phenotype in UC mice. Subsequently, the HPLC-Q-Exactive-MS technology was used to analyze the serum migration components of TFLQW, and 20 serum migration components were identified through spectral library searching and mass spectrometry identification processes. The Swiss Target Prediction database was used to predict the target of the components, and UC targets were searched in disease databases GeneCards, OMIM, and DisGeNet. Venny 2.1.0 software was used to draw a Venn diagram to obtain 113 intersection targets. The "drug-components-targets-disease" network was constructed using Cytoscape 3.8.0 software, and the Centiscape 2.2 was used to screen for core components. Components such as palmatine, berberine, jatrorhizine, coptisine, chrysophanol, and resveratrol in TFLQW may be the active substances exerting the therapeutic effect. The String database was used to analyze protein-protein interaction (PPI) and to screen for key targets, suggesting that TFLQW exerts the therapeutic effect on UC by targeting STAT3, PIK3R1, SRC, PIK3CA, MAPK1, and AKT1, among others. The David database was used for GO enrichment analysis and KEGG pathway analysis of the intersection targets, and revealed that the pathways involved in TFLQW treatment of UC mainly include cancer, PI3K/AKT and C-type lectin receptor signaling pathways. Molecular docking was performed on key targets and core components, and PIK3CA binded well with various active components. Western blot was used to detect the protein expression of PI3K/AKT signaling pathway in mouse colon tissue. This study has confirmed the therapeutic effect of TFLQW on UC and preliminarily identified the potential active components and signaling pathways of TFLQW in treating UC. These findings provide a reference for in-depth research into the material basis and action mechanisms of the compound preparation, and also offer a theoretical basis for the clinical application of TFLQW. The animal welfare and experimental procedures were approved by the Medical Ethics Committee of Inner Mongolia Medical University (ethics approval number: YKD202302093).