Gene deletion analysis is a powerful tool for resolving the contributions of individual open reading frames to the physiology of cells. Analysis of deletion phenotypes in conjunction with a specific pathway reporter can identify constituents of a physiological pathway and reveal potential effectors that regulate the pathway by quantifying the phenotypic responses of the mutant cells. This article describes a high-throughput method of analyzing a yeast gene deletion library for novel G-protein signaling modulators using a yeast pheromone pathway-specific reporter.