The in vitro micronucleus test is currently used as a screening assay during the early stages of drug development by pharmaceutical companies to identify chemicals likely to produce positive outcomes in the in vitro chromosome aberration assay. For several reasons the assay is being considered as an alternative to the aberration assay—it requires less laboratory time, less material and less training. However, the current screening protocols are not rigorous enough to fully satisfy concerns about genotoxic safety. Using a protocol previously developed by testing 16 chemicals, this manuscript contributes to the validation of the protocol using 10 additional chemicals. Furthermore, conclusions drawn from the developmental effort regarding the need for an extended exposure in the absence of metabolic activation, the number of cells to be counted, and the preferred statistical procedure for the assay are re-examined. The recommended, validated protocol utilizes cytochalasin B and 4h exposures in the presence and in the absence of metabolic activation, specifies the need to test to a relative survival rate of approximately 50%, requires the counting of 2000 binucleated cells per treatment concentration, and employs a trend test for statistical analysis of the data.