Eosinophils are multifunctional leukocytes classically described as being involved in helminth parasitic infections and allergic diseases. Previously restricted to an exclusive role in the release of cytotoxic mediators, they are now also considered to be immunoregulatory cells and potential effectors in innate immune responses. Eosinophils are mainly found in tissues, so specific procedures are needed for their isolation from venous blood and for functional assays. Murine models are very useful for the dissection of eosinophil physiology in vivo. But murine eosinophils significantly differ from human ones. A complete understanding of eosinophil biology therefore requires comparative study of eosinophils from different mammalian species. We summarize here the main experimental protocols used to study human, mouse, and rat eosinophil biology. We focus on technical improvements of existing methods that optimize purification and in vitro functional studies of eosinophils.