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A new approach for proteome-wide profiling drug binding proteins by using monolithic capillary affinity chromatography in combination with HPLC–MS/MS is reported.Two immunosuppresive drugs,namely FK506 and cyclosporin A,were utilized as the experimental models for proof-of-concept.The monolithic capillary affinity columns were prepared through a single-step copolymerization of the drug derivatives with glycidyl methacrylate and ethylene dimethacrylate.The capillary chromatography with the affinity monolithic column facilitates the purification of the drug binding proteins from the cell lysate.By combining the capillary affinity column purification and the shot-gun proteomic analysis,totally 33 FK506-and 32 CsAbinding proteins including all the literature reported target proteins of these two drugs were identified.Among them,two proteins,namely voltage-dependent anion-selective channel protein 1 and serine/threonine-protein phosphatase PGAM5 were verified by using the recombinant proteins.The result supports that the monolithic capillary affinity chromatography is likely to become a valuable tool for profiling of binding proteins of small molecular drugs as well as bioactive compounds.