A high-resolution screening platform, coupling online affinity detection for mammalian cytochrome P450s (Cyt P450s) to gradient reversed-phase high-performance liquid chromatography (HPLC), is described. To this end, the onlineCyt P450 enzyme affinity detection (EAD) system was optimized for enzyme (β-NF-induced rat liver microsomes), probe substrate (ethoxyresorufine), and organic modifier (methanol or acetonitrile). The optimized Cyt P450 EAD system has first been evaluated in a flow injection analysis (FIA) mode with 7 known ligands of Cyt P450 1A1/1A2 (β-naphthoflavone, ßnaphthoflavone, ellipticine, 9-hydroxy-ellipticine, fluvoxamine, caffein, and phenacetin). Subsequently, IC 50 valueswere online in FIA-mode determined and compared with those obtained with standardmicrosomal assay conditions. The IC 50 values obtained with the online Cyt P450 EAD system agreed well with the IC 50 values obtained in the standard assays. For highaffinity ligands ofCyt P450 1A1/1A2, detection limits of 1 to 3 pmol injected (n= 3; signal to noise [S/N] = 3) were obtained. The individual inhibitory properties of ligands in mixtures of the ligands were subsequently investigated using an optimized Cyt P450 EAD system online coupled to gradient HPLC. Using the integrated online gradient HPLC Cyt P450 EAD platform, detection limits of 10 to 25 pmol injected (n= 1; S/N= 3) were obtained for high-affinity ligands. It is concluded that this novel screening technology offers new perspectives for rapid and sensitive screening of individual compounds in mixtures exhibiting affinity for liver microsomal Cyt P450s.