Efficient and targeted screening and isolation of bioactive compounds from complex natural products is still a challenging work. Herein, diagnostic ion filtering based high‐performance liquid chromatography–quadrupole time‐of‐flight−tandem mass spectrometry was firstly developed to screen six main iridoid glycosides from Hedyotis diffusa. Then, online extraction‒high‐speed counter current chromatography was proposed for targeted enrichment and preparative isolation using ethyl acetate/n‐butanol/water (4.5:0.5:5, v/v/v) as solvent system. After that, Sephadex LH‐20 column chromatography using methanol as solvent system was selected for further purification of six iridoid glycosides with purities over 98%. They were finally identified as monotropein, desacetylasperuloside acid, asperuloside, 6‐O‐(Z)‐p‐coumaroyl scandoside methyl ester, 6‐O‐(Z)‐feruloyl scandoside methyl ester, and 6‐O‐(E)‐p‐coumaroyl scandoside methyl ester. And their anti‐inflammatory activities were evaluated and confirmed by lipopolysaccharide activated RAW 264.7 macrophages. Obviously, the results provide a scientific basis for the potential applications of H. diffusa, and the developed methodology is efficient and reliable for targeted screening and isolation of bioactive compounds from natural products.