Background. This study investigated the pharmacological mechanisms of Tong-Xie-Yao-Fang (TXYF) against irritable bowel syndrome (IBS). Methods. The chemical profile of TXYF was identified through UHPLC-QTOF-MS. Next, the obtained chemical profile served as the basis for network pharmacological analysis. Finally, the predictive performance of network pharmacology was validated by conducting molecular docking and animal experiment. Results. Seven key compounds of TXYF, namely, quercetin, ellagic acid, nobiletin, formononetin, isorhamnetin, vestitol, and licochalcone, were confirmed as the key components acting on IBS. TXYF treatment on IBS was mainly realized through the regulation of some key pathways of immune system, such as inflammatory bowel disease, cytokine-cytokine receptor interaction, HIF-1, and T cell receptor signaling pathway. NOS2, ACHE, ESR1, PTGS2, and RELA were the target genes of TXYF to improve IBS. Stable bonds between the key components and the core target genes were further verified by the results of molecular docking. In vivo experiments confirmed the effects of TXYF on IBS. Further Western blot analysis showed that NOS2, ACHE, and ER alpha were significantly upregulated in the model group in comparison with controls (P<0.001) but then significantly downregulated after treatment with TXYF for 14 days (P<0.001). Conclusion. This study applied an integrated method based on network pharmacology and experimental validation to examine the underlying "multicomponent, multitarget, and multipathway" mode of action of TXYF in treating IBS. The current findings provided indicative paradigms and new insights into exploring the multitarget therapeutic mechanism of Chinese herbal compound.