A simple and flexible setup for conducting drug metabolism studies is described in this report. A heating block was designed for the Multimek liquid handler platform for incubation of multiple samples at 37°C in a 96-well format. This setup enables the rapid performance of drug metabolism experiments on a large number of samples. In this report, the authors present the validation of the system by 1) showing reproducible and consistent determination of the in vitro half-life of midazolam in every well across the entire plate and 2) determination of metabolic parameter values of midazolam, testosterone, diclofenac, warfarin, and dextromethorphan and inhibition parameter values of quinidine and ketoconazole, all comparable to literature values. In addition, the authors demonstrate the application of the setup to determining the metabolic stability of a set of proprietary compounds, the inhibition of activity of cytochrome P450 (CYP) enzymes, and the conduct of a single combination experiment that can simultaneously determine the metabolic stability and CYP inhibition activity. Overall, the system represents a simple, high-throughput and useful tool for drug metabolism screening in drug discovery. (Journal of Biomolecular Screening 2003:544-554) Key words: drug metabolism, enzyme kinetics, high-throughput screening, cytochrome P450 inhibition INTRODUCTION IN DRUG DISCOVERY, high-throughput screening (HTS) assays provide low-resolution data for preliminary assessment of the desired properties of the compounds. One of the properties-interaction with the liver enzymes, mainly the cytochrome P450s (CYP)-has become as important for the preliminary assessment of the compounds as assessing their selectivity and sensitivity toward the target. It is a liability if a compound is rapidly metabolized or is an inhibitor of the enzymes, and knowledge of these liabilities early in a drug discovery program can be used to either rectify the problem or eliminate a compound from development. Thus, there is a continuing effort to devise ways to assess these properties rapidly and in valid experimental setups.Various attempts have been made to increase the throughput of these assays. For example, fluorescence-based assays for determining the inhibition of CYP activity are available for a number of isozymes and are being used in HTS of small molecules. 1 In these assays, the nonfluorescent substrate, upon enzymatic conversion, generates a fluorescent metabolite that is detected in a fluorescence plate reader. The assay is often performed at ambient temperature with single endpoint measurement of the reaction to make it easy to adapt to robotic systems. This method can be applied to determining IC 50 values, although at a relatively low throughput. The most serious drawback of this method is the interference from the intrinsic fluorescence or possible fluorescence-quenching effects of compounds with the detection of substrate metabolite. Another issue is that the nonfluorescent substrates that convert into sensitive fluorescent metab...