Abstract With the recent increase in targeted therapies for the treatment of eosinophil-associated disorders, the availability of animal models that mimic human eosinophil responses is a priority. Although murine models have been useful in this regard, differences in eosinophil biology and pathogenesis between humans and mice have limited their utility in some settings. Rhesus macaques provide a potential solution to this problem. Multi-parameter flow cytometry was used to compare the surface phenotype of human and rhesus macaque eosinophils in whole blood. Based on the findings, a strategy for magnetic bead purification of rhesus eosinophils was devised. Activation of purified eosinophils was assessed by surface staining with anti-CD69 antibody, and function was evaluated in a standard chemotaxis assay. Similar to human eosinophils, rhesus macaque eosinophils were found to express EPX and functional CCR3. Siglec-8 expression was not detected. Rhesus neutrophils do not express CD16, the surface marker most commonly used for eosinophil purification in humans. Purity was established by counting 300 cells on cytospin slides stained with Diff-Quik and by flow cytometry. Rhesus eosinophils were isolated from six Rhesus monkeys by Ficoll sedimentation followed by immunomagnetic negative selection using anti-CD64 antibody. Purity of >90% was achieved in 2 animals. Purified eosinophils were not activated, as assessed by CD69 expression, and chemotaxis to eotaxin was preserved. These data provide a comprehensive comparison of the surface expression patterns of human and rhesus granulocytes and a method for purification of viable and functional rhesus eosinophils for in vitro studies.