We developed an In Vivo Liver Micronucleus method that uses automated flow cytometry to obtain this important chromosome damage endpoint and combined it with a 3Rs friendly study design. The experimental design not only examines chromosome damage in hepatocytes, but also in red blood cells, and additionally included our Pig-a gene mutation endpoint. This approach is also meant to be very resource efficient such that exposures only occur three times during a standard work week and no work is required over the weekend. We applied this design for 1 or three weeks and studied 13 genotoxicants with varied activities. In addition to demonstrating the efficiencies that can be achieved by combining several genotoxicity assays into a single study, we demonstrated the value of examining the complementary endpoints.  Every compound was positive in at least one assay, several in more than one. However, not all assays were able to detect every single agent, thus suggesting that comprehensive characterization of a compounds’ genotoxicity requires the use of multiple assays to cover various activities and sites of action. Use of these assays in our optimized study design can lead to better interpretation of potential hazard and does so in a way that maximizes the amount of information that can be achieved in a single study. This eliminates the need to perform several independent studies and reduces and refines overall animal use.