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The emergence of new pathogens, such as Middle East respiratory syndrome coronavirus (MERS-CoV), poses serious challenges to global public health and highlights the urgent need for methods to rapidly identify and characterize potential therapeutic or prevention options, such as neutralizing antibodies. Spike (S) proteins are present on the surface of MERS-CoV virions and mediate viral entry. S is the primary target for MERS-CoV vaccine and antibody development, and it has become increasingly important to understand MERS-CoV antibody binding specificity and function. Commonly used serological methods like ELISA, biolayer interferometry, and flow cytometry are informative, but limited. Here, we demonstrate a high-throughput protein binding inhibition assay using image cytometry. The image cytometry-based high-throughput screening method was developed by selecting a cell type with high DPP4 expression and defining optimal seeding density and protein binding conditions. The ability of monoclonal antibodies to inhibit MERS-CoV S binding was then tested. Binding inhibition results were comparable with those described in previous literature for MERS-CoV spike monomer and showed similar patterns as neutralization results. The coefficient of variation (CV) of our cell-based assay was <10%. The proposed image cytometry method provides an efficient approach for characterizing potential therapeutic antibodies for combating MERS-CoV that compares favorably with current methods. The ability to rapidly determine direct antibody binding to host cells in a high-throughput manner can be applied to study other pathogen-antibody interactions and thus can impact future research on viral pathogens., (Published by Elsevier B.V.)