Screening diagnostic biomarkers can be challenging due to the complexity of traditional Chinese medicine (TCM) and ambiguous pharmacological mechanisms. In this study, we reported an integrated strategy for accurately screening diagnostic biomarkers based on metabolomics coupled with network pharmacology. First, a feasible pharmacological model was established through systems pharmacology and based on metabolomicsbased techniques to explore diagnostic biomarkers. While the components satisfying the q-value < 0.05, fold change (FC) >= 1.2 or FC <= 0.8, coefficient of variance (CV) <= 30%(QC) and the variable importance in the project (VIP) value > 1 are considered to be diagnostic biomarkers. Second, the ingredients were retained only when oral bioavailability (OB), Caco-2 permeability, drug half-life, TPSA and drug likeness (DL) satisfied the criteria (OB >= 40%; Caco-2 >= -0.4; HL >= 4 h; TPSA(<)140; DL >= 0.18) suggested by the TCMSP database. Moreover, ingredients that exhibit extensive biological activity in TCM are also retained. Third, the effect targets of TCM were screened using the TCMSP database, Swiss Target Prediction and STICH online software. Disease targets were gathered from the therapeutic target database (TTD), PharmGkb and TCMSP database. Hub genes were screened by potential protein-protein interaction (PPI) network pharmacology analysis. Finally, a metabolic network pathway is established between the diagnostic biomarker and the hub gene. In the network analysis of metabolic pathways, most of the genes involved in this pathway are the second-step-obtained hub genes, which can explain the accuracy of the identified biomarkers. The proposed integrated strategy was successfully applied to explore the mechanism of action of Pulsatilla decoction (PD) in the treatment of acute ulcerative colitis (UC). Based on this integrated strategy, 23 potential biomarkers of acute UC treated with PD were identified. In conclusion, the integrated strategy provides novel insights into network pharmacology and metabolomics as effective tools to illuminate the mechanism of action of TCM.