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Meconopsis integrifolia (Maxim.) Franch. is used extensively in traditional Tibetan medicine for its potent anti‐inflammatory properties. In this study, six cyclooxygenase‐2 (COX‐2) inhibitors were purified from M. integrifolia using high‐speed counter‐current chromatography guided by ultrafiltration liquid chromatography (ultrafiltration‐LC). First, ultrafiltration‐LC was performed to profile the COX‐2 inhibitors in M. integrifolia. The reflux extraction conditions were further optimized using response surface methodology, and the results showed that the targeted COX‐2 inhibitors could be well enriched under the optimized extraction conditions. Then the six target COX‐2 inhibitors were separated by high‐speed countercurrent chromatography with a solvent system composed of ethyl acetate/n‐butanol/water (4:1:4, v/v/v. Finally, the six COX‐2 inhibitors, including 21.2 mg of 8‐hydroxyluteolin 7‐sophoroside, 29.6 mg of 8‐hydroxyluteolin 7‐[6′''‐acetylallosyl‐(1→2)‐glucoside], 42.5 mg of Sinocrassoside D3, 54.1 mg of Hypolaetin 7‐[6′′′‐acetylallosyll‐(l→2)‐3′′‐acetylglucoside, 30.6 mg of Hypolaetin 7‐[6′′′‐acetylallosyll‐(l→2)‐6′′‐acetylglucoside and 17.8 mg of Hypolaetin were obtained from 500 mg of sample. Their structures were elucidated by 1H‐NMR spectroscopy. This study reveals that ultrafiltration‐LC combined with high‐speed counter‐current chromatography is a robust and efficient strategy for target‐guided isolation and purification of bioactive molecules. It also enhances the scientific understanding of the anti‐inflammatory properties of M. integrifolia but also paves the way for its further medicinal applications. [ABSTRACT FROM AUTHOR]