Rapid drug metabolite profiling can be achieved using fast chromatographic separation and fast mass spectrometric scanning without compromising the separation efficiency. Fast chromatographic separations of drug and its metabolites can be achieved by eluting from a short narrow-bore guard cartridge column (20 x 2 mm I.D., 3 microns BDS Hypersil C8) at flow-rate of 1.0 ml/min and with a gradient volume greater than 90 column volumes. The need for chromatographic separation is important for automated data dependent multiple-stage mass spectrometry (MSn) experimentation. The total analysis time of 8 min permits profiling of metabolites in a 96-well plate in 13 h. The narrow chromatographic peaks resulting from the high flow-rate require the use of a mass spectrometer capable of fast scan speed due to the need to perform multiple MS experiments within the same chromatographic analysis. A method has been developed for screening potentially biologically active in vitro microsomal metabolites by affinity binding with a receptor. After separation by centrifugal ultrafiltration, the bound ligands are released and characterized by LC-MS. In vitro microsomal metabolites of tamoxifen, raloxifene and adatanserin were screened for potential biological activity using this method. The in vitro metabolites of tamoxifen captured by the receptor include N-demethyltamoxifen and three species of hydroxytamoxifen; these data are consistent with those from a conventional binding study and bioassay. In addition, both hydroxyraloxifene and dihydroxyraloxifene are also recognized by the receptor. The specificity of the molecular recognition process is illustrated by the absence of binding with control microsomal incubate and with adatanserin and its metabolites. Therefore, active metabolites can be rapidly profiled by fast LC, automated MSn, and receptor binding. This information can be obtained quickly and can add value to the drug discovery process.