Abstract: Urtica fissa, a commonly used traditional Chinese herb, frequently causes contact dermatitis during application. However, the inflammatory components have not been reported. In this study, RAW264.7 cell model was employed first to identify the dichloromethane fraction as the primary inflammatory fraction. Then, using chromatographic techniques such as silica gel column chromatography and preparative HPLC, combined with HR-MS, NMR, and UPLC-IM-Q-TOF-MS analysis, 44 compounds, including nettlol, cinnamic acid, and salicylic acid, were identified from the dichloromethane fraction. Subsequently, the potential inflammatory components were confirmed by transcriptomic analysis and molecular docking. Finally, the inflammatory effects of these candidates were evaluated by an open topical application test on mouse skin. The results showed that cinnamic acid and salicylic acid significantly induced skin erythema and ear swelling in mice, and markedly elevated histamine and NO levels in serum and skin, confirming them as the key inflammatory constituents. All animal experiments were reviewed and approved by the Ethics Committee of Shanghai Jiao Tong University Laboratory Center (A2025060-001). The findings of this study provided a scientific basis for further research and application of U. fissa.