Aggregated compounds can promiscuously and nonspecifically associate with proteins resulting in either false inhibition or activation of many different protein target classes. We developed a high-content imaging assay in a 384-well format using fluorescently labeled target proteins and an Operetta cell imager to screen for compound aggregates that interact with target proteins. The high-throughput assay can not only directly detect the interaction between compound aggregators and the target of interest, but also determine the critical aggregation concentration (CAC) of a given promiscuous small molecule.