Ensemble docking is now commonly used in early-stage in silico drug discovery and can be used to attack difficult problems such as finding lead compounds which can disrupt protein?protein interactions. We give an example of this methodology here, as applied to fibroblast growth factor 23 (FGF23), a protein hormone that is responsible for regulating phosphate homeostasis. The first small-molecule antagonists of FGF23 were recently discovered by combining ensemble docking with extensive experimental target validation data (Science Signaling, 9, 2016, ra113). Here, we provide a detailed account of how ensemble-based high-throughput virtual screening was used to identify the antagonist compounds discovered in reference (Science Signaling, 9, 2016, ra113). Moreover, we perform further calculations, redocking those antagonist compounds identified in reference (Science Signaling, 9, 2016, ra113) that performed well on drug-likeness filters, to predict possible binding regions. These predicted binding modes are rescored with the molecular mechanics Poisson?Boltzmann surface area (MM/PBSA) approach to calculate the most likely binding site. Furthermore, our findings suggest that the antagonist compounds antagonize FGF23 through the disruption of protein?protein interactions between FGF23 and fibroblast growth factor receptor (FGFR).