The need to segregate poultry based on sex is driven by sex-related differences in growth rate, market age, management practices, and nutritional requirements. Each day, poultry industry staff globally would ideally like to determine the sex of >150 million newly hatched birds. Currently, this can be done only manually at the hatchery, which is a virtually impossible undertaking. It is becoming more difficult each year to conduct manual sexing because this skill is disappearing from the workforce, is becoming unaffordable to the industry, and is encumbered by such negative effects as repetitive motion disorder. Automated sex sorting of eggs before hatching could resolve many, if not all, of these problems. We have developed a facile, rapid, and low-cost yeast-based assay that distinguishes male from female embryonated eggs before hatching based on the estrogen concentration of their allantoic fluid. Herein, we describe this novel sex-sorting technology, which we believe offers the potential to standardize and automate sex sorting in the poultry industry.