Screening of monoclonal antibodies for binding specificity and selectivity has been attempted using multiple methods. Flow cytometric analysis is rapid and non-subjective. These studies used flow cytometry to screen antibodies for their antigen specificity and intensity of fluorescent signal to cells expressing antigens from Venezuelan, Western, and Eastern (North American and South American) equine encephalitis viruses. Avian cells were infected with each of the strains, fixed and stained with primary antibodies and fluorescent conjugate. Four antibodies were chosen as most suitable to advance to quantitative testing on the basis of differential specificity, one for each viral strain. Three trials of seven replicates were used to establish that these antibodies generated reproducible and consistent signal that discriminate cells infected with each specific virus relative to uninfected cells. These flow cytometry measurements have demonstrated the essential specificity, selectivity and reproducibility of four antibodies for further development of assays to support vaccine production.