Several intracellular allosteric CCR2 antagonists, including biaryl sulfonamides (e.g. SD-24 (1) and 2) and pyrrolones (e.g. CCR2-RA (4)) have already been identified and chemically optimized in the absence of any detailed structural information on intracellular CCR2 antagonism (Chart 1). With the disclosure of the crystal structure of CCR2 in complex with the CCR2-RA eutomer 4-[R], Zheng et al. laid the foundation for the structure-based development of further intracellular CCR2 antagonists, such as the covalent biaryl sulfonamide analogue 3. Besides structural insights on receptor-ligand interactions, molecular tools that enable an unambiguous identification and characterization of intracellular CCR2 ligands are of utmost importance for drug discovery campaigns in this field. With the radioligand [ 3 H]CCR2-RA, Zweemer et al. have therefore reported a highly valuable molecular tool that can be utilized to detect binding to the IABS of CCR2. Chart 1. Chemical structures of selected intracellular allosteric antagonist (1-5). Biological activity (Ki values) as reported by a Peace et al., Wang et al., In more recent studies, the eutomer [ 3 H]CCR2-RA-[R] was used instead of the racemic mixture. This molecular tool has frequently been applied and has successfully guided the structure-based development of CCR2 antagonists such as 3 and 5. However, radioligand binding assays are accompanied by several drawbacks, such as high infrastructure requirements according to radiation protection measures, the production of radioactive waste, and a laborious (heterogeneous) assay protocol including washing steps, to remove the unbound radioligand prior to assay readout. The latter is also the reason why radioligand binding assays are often not well-suited for a continuous readout or the detection of low affinity binders, such as fragments. Recently, we reported the development of a fluorescent tracer targeting the IABS of CCR9, which was successfully applied for binding studies using the non-isotopic NanoBRET technology. Herein, we aimed at developing a fluorescently labeled intracellular CCR2 ligand, in order to provide a molecular tool that allows to study direct binding to the IABS of CCR2 in a non-isotopic manner.