BACKGROUNDYinhuang Hanhua dropping pill (YHHHDP) is a Chinese herbal formula that is commonly used in medical practice for managing of acute pharyngitis (AP) and other upper respiratory tract infections. However, the active components, molecular targets, and mechanisms underlying its therapeutic effects remain poorly understood.PURPOSEThe objective of this study was to identify the active components and molecular mechanisms of YHHHDP in the therapy of AP.METHODSThe efficacy of YHHHDP was assessed in an ammonia-induced AP rat model and LPS-stimulated RAW264.7 macrophages based on HE staining, immunofluorescence, ELISA, and qRT-PCR. The active components, molecular targets, and pathways were investigated using integrating compositional analysis, metabolomics, and network pharmacological analysis. Furthermore, the interactions between active components and targets, as well as the key signaling pathways were verified using Western blotting (WB), Bio-layer interferometry(BLI), cellular thermal shift assay (CETSA), etc. RESULTS: YHHHDP significantly ameliorated the pathological damage and inflammatory response in the pharyngeal tissues of AP rats. YHHHDP also inhibited LPS-induced expression of pro-inflammatory factors in RAW264.7 cells. Research involving component analysis, network pharmacology, and metabolomics showed that baicalin, chlorogenic acid, and 10 other compounds are the active components of YHHHDP used in AP treatment. The core targets of these active ingredients were TNF-α, AKT1, and COX-2. The mechanism of action primarily involved the PI3K-AKT/NF-κB/MAPK signaling pathway. Tryptophan and arachidonic acid metabolism were the primary metabolic pathways. Meanwhile, animal and Cellular verification experiments demonstrated that the active ingredients of YHHHDP can stably bind to key target proteins, thereby regulating key metabolites in tryptophan and arachidonic acid metabolism, inhibiting the expression of key proteins in the PI3K-AKT/NF-κB/MAPK signaling pathway, reducing the release of pro-inflammatory factors, and improving the symptoms of AP.CONCLUSIONThis study revealed that 10 components, such as baicalin and luteolin, are the active ingredients of YHHHDP in AP treatment. The mechanism of action involves the inhibition of TNF-α, AKT1, and COX-2, thereby exerting therapeutic effects through the regulation of arachidonic acid and tryptophan metabolism and the inhibition of the PI3K-AKT/NF-κB/MAPK signaling pathway. This study provided an invaluable scientific basis for the clinical application and development of YHHHDP.