This article describes the first step toward full (that includes conditions for both absence and presence of metabolic activation) validation and drug discovery application of a 96-well, automated, high-content micronucleus (HCMN) assay. The current validation tests were performed using Chinese hamster ovary cells, in the absence of metabolic activation, against three distinct sets of drug-like compounds that represent all stages of a drug discovery pipeline. A compound categorization scheme was created based on quantitative relationships between micronucleus (MN) signals, cytotoxicity, and compound solubility. Results from initial validation compounds (n = 38) set the stage for differentiating overall positive and negative MN inducers. To delve deeper into the compound categorization process, a more extensive validation set, consisting of a larger set (n = 370) of "drug-like but less optimized" early-stage compounds, was used for further refinement of positive and negative compound categories. The predictivity and applicability of the assay for clinical stage compounds was ascertained using (n = 168) clinically developed marketed drugs or well-studied compounds. Upon full validation, a detailed analysis of results established five compound categories--NEG (negative), NEG/xx μM (negative up to the solubility limit of xx μM), WPOS (weak positive), POS (positive), and INCON (inconclusive). Furthermore, examples of lead-finding applications and ongoing investigative HCMN activities are described. A proposal is offered on how the HCMN assay can be positioned in parallel to the overall stage gates (e.g., scaffold selection, lead optimization, late-stage preclinical development) of drug discovery programs. Because of its greater throughput, 1-week turnaround time, and a substantially reduced (1-2 mg) requirement for compound consumption, the HCMN assay is appropriate for developing structure-genotoxicity relationships and for mechanistic genotoxicity studies. The assay does not replace the Organization for Economic Cooperation and Development-compliant, non-good laboratory practice in vitro MN test (e.g., slide-based MN test in TK6 lymphoblastoid cells) that is used for full characterization of lead candidates.