Abstract Background: Docking sites on mitogen-activated protein kinases (MAPKs) direct protein binding interactions and generate signal specificity. Therapeutically targeting these sites in cancer offers a way to potentially overcome many issues associated with ATP-competitive MAPK inhibitor design, such as high cellular ATP concentrations and highly conserved active sites among MAPKs. Extracellular signal-regulated kinases 1/2 (ERK1/2) are MAPKs that have a pro-tumorigenic role in numerous cancers and possess two protein docking sites that are distinct from the active site: the D-recruitment site (DRS) and F-recruitment site (FRS). In this study, we have developed a high-throughput fluorescence anisotropy screening to identify small molecule inhibitors that target the DRS of ERK. Methods: DRS inhibitors were detected in the high-throughput screening by using fluorescence anisotropy to measure the ability of over 22,000 molecules to competitively displace a fluorescent peptide from the DRS of inactive ERK2. The top 100 compounds from this screening were validated with a secondary radioactive kinase assay. In this assay, the ability of each compound to prevent the phosphorylation of a DRS-specific peptide by active ERK2 was assessed using [γ-32P]-labeled ATP substrate to measure enzyme activity. The top nine compounds were characterized by in vitro dose-response assays against wild-type and C159S ERK2, a DRS mutant. Inhibition of ERK2 phosphorylation of an FRS substrate and JNK2 phosphorylation of c-Jun was evaluated in vitro to determine compound specificity. The potency and specificity of the compounds were also assessed in EGF-stimulated HEK293T cells for their ability to prevent ERK1/2, JNK1/2, and p38α phosphorylation using Western blot. The compounds were tested for irreversible inhibition in vitro through ERK activity tests after exposure to, and subsequent removal of the compounds. Results: Of the nine hit compounds found from the screening, four reversibly inhibited the DRS of ERK2 with in vitro IC50 and Ki values under 5 μM, and did not show significant inhibition of the FRS or C159S ERK2. Two of these compounds also reduced ERK activation to basal levels in MAPK pathway-stimulated HEK293T cells at concentrations of 5 μM. However, in specificity tests these inhibitors demonstrate ability to also target JNK2 and p38α. Conclusions: The fluorescence anisotropy screening developed here can be used to identify potent inhibitors that target the DRS of ERK. These inhibitors can be used to probe ERK signaling events that are mediated by the DRS and can be further optimized to yield highly selective ERK DRS inhibitors with potential therapeutic value. Citation Format: Rachel M. Sammons, Tamer S. Kaoud, Ashwini K. Devkota, Eun J. Cho, Kevin N. Dalby. A high-throughput fluorescence anisotropy screening for discovery of inhibitors that target the D-recruitment site of ERK in vitro and in cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3648. doi:10.1158/1538-7445.AM2015-3648