Previously, this laboratory reported on the development of a flow cytometry-based method that automates the assessment of the mitotic index (MI) and numerical chromosome changes in chemically treated cultures of human lymphocytes [Muehlbauer PA and Schuler MJ, 2003, 2005]. With this method, testing design can easily include numerous well-spaced doses to better define the shape of MI dose response curves. In addition, the hypodiploid, hyperdiploid, and polyploid mitotic populations are available simultaneously to determine the biological relevance of polyploidy effects during the conduct of the assay. The current work describes the integration of this flow cytometry-based method into the routine conduct of good laboratory practice structural chromosome aberration assays in vitro, and discusses improvements in evaluating cytotoxicity and polyploidy endpoints. Additional methods for simultaneous assessment of cell death (sub-G1 DNA) are shown in combination with the MI to provide a more complete evaluation of cytotoxic conditions. A total of 30 pharmaceutical compounds were assayed in compliance with Organization for Economic Cooperation and Development and International Conference on Harmonization guidelines. The inclusion of numerous well-spaced doses improved high dose selection and resulted in fewer high dose artifacts. Only 1 compound in 30 produced a positive response in structural aberrations. In comparison, polyploidy induction was observed in 22 of 30 (73%) compounds, with no apparent increases in numerical chromosomal aberrations. These studies show that flow cytometry-based methods can be used to better characterize cytotoxicity dose-response relationships and improve the detection of aneugens.