Abstract: High-throughput screening (HTS) assays have been utilized extensively for evaluation of pharmacological and toxicological effects of chemicals and drugs. However, all these assays, no matter cell-based or enzyme-based, lack metabolic capacity for chemicals. Thus, results from these assays may not accurately reflect in vivo activity/toxicity of a compound. To better correlate in vivo activity of compounds, we added a metabolic component (e.g., human liver microsomes) into a HTS assay that has been used to determine the activities of acetylcholinesterase inhibitors. We found that the activities of acetylcholinesterase inhibitors determined by this new assay system are closer to those reported by in vivo assays. Our data demonstrated that incorporating drug metabolic capability into in vitro HTS assays produces more physiologically relevant in vitro assays that can accurately measure compound activity.