Eosinophils are bone marrow-derived cells that differentiate in the bone marrow and migrate into the peripheral blood primarily under the regulation of interleukin (IL)-5. Despite the fact that eosinophils have been mostly studied in the context of allergic inflammatory diseases, eosinophils accumulate in multiple tumors. In fact, recent data highlight key anti-tumorigenic activities for eosinophils. Thus, developing simple assays that will dissect the interactions between eosinophils and tumor cells is important since these assays will provide tools to study eosinophils in the tumor microenvironment. In this chapter, we provide detailed methods for isolating eosinophils from Il5 transgenic mice. Furthermore, we provide methodology to assess eosinophil chemotaxis in response to tumor-secreted factors. Finally, we describe a co-culture system of eosinophils and tumor cells aimed to determine the cytotoxic capabilities of eosinophils.