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A strategy based on chemical markers’ fishing and knockout has been proposed for holistic activity and interaction evaluation of the bioactive components in herbal medicines (HMs).It was devised to screen bioactive-compound group that represents the efficacy of HM,estimate the bioactivity contribution of each component and elucidate the interactions of multi-components.This strategy was accomplished through the following steps: (1) screen out the chemical markers (target peaks) in a HM fingerprint using online two-dimensional turbulent flow chromatography/liquid chromatography–mass spectrometry technique,(2) fish target peaks and knockout any interested peak,and (3) evaluate the bioactivities of fishing and knockout portions.After comparison of the bioactivities of samples containing different target peaks,the efficacy of target-peak group,bioactivity contribution of each compound,and the interactions of multi-components are elucidated.Using Acetylcholinesterase (AChE) and Bulbs of Lycoris radiata (L.Herit.) Herb.(BLR) as the experimental materials,four target peaks were screened out as the AChE binders.By target peaks’ fishing and knockout,combined with activity evaluation,we observed that the bioactivity of the four-peak mixture is similar with the global bioactivity of BLR extract,and there are significant suppressive actions among these four target peaks.These results indicate that this proposed strategy is a useful approach for holistic screening of bioactive-compound group and elucidation of the multi-component interactions in HM.