There are a variety of methods available for determining ethanol tolerance phenotypes in Saccharomyces cerevisiae. Many of these methods are limited in throughput and/or application. We describe here a microtiter-plate, growth-based, liquid-culture, rapid ethanol tolerance assay (RETA) that overcomes these limitations. Determinations of ethanol tolerance gave results that were comparable to shake-flask cultures, and there was a high level of intra-and inter-plate reproducibility. We report the successful application of this assay to determining the segregation pattern of ethanol tolerance in backcrosses of two ethanol-tolerant mutants (SM and CM) to a non-tolerant parent (W303-1A); RETA was used to determine ethanol tolerance in numerous progeny resulting from these crosses. This assay, or variations thereof, will prove be of great value for anyone attempting to develop quantitative, high-throughput growth assays for yeast or other microorganisms.