Environmental conditions experienced during early larval stages dictate the developmental trajectory of the nematode C. elegans. Favorable conditions such as low population density, abundant food, and lower temperatures allow reproductive growth, while stressful conditions promote entry of second-stage (L2) larvae into the alternate dauer developmental stage. Population density is signaled by the concentration and composition of a complex mixture of small molecules that is produced by all stages of animals, and is collectively referred to as dauer pheromone; pheromone concentration is a major trigger for dauer formation. Here, we describe a quantitative dauer formation assay that provides a measure of the potency of single or mixtures of pheromone components in regulating this critical developmental decision.