Peptide aptamers have primarily been used as research tools to manipulate protein function and study regulatory networks. However, they also find multiple applications in therapeutic research, from target identification and validation to drug discovery. Because of their unbiased combinatorial nature, peptide aptamers interrogate the biological significance of numerous molecular surfaces on target proteins. Their use enables the identification and validation of some of these surfaces as interesting therapeutic targets to pursue. Peptide aptamers can subsequently be used to guide the discovery of small molecule drugs specific for these molecular surfaces.Here, we present a high-throughput screening assay that identifies small molecules that displace interactions between proteins and their cognate peptide aptamers. AptaScreen is a duplex yeast two-hybrid assay featuring two luciferase reporter genes. It can be performed in 96- or 384-well plates and can be fully automated.