Although tumor-associated macrophages are generally immunosuppressive, macrophages may also promote tumor clearance via phagocytosis of live tumor cells. Here, we present a protocol for assessing macrophage engulfment of tumor cells in vitro using flow cytometry. We describe steps for cell preparation, reseeding macrophages, and setting up phagocytosis. We then detail procedures for collecting samples, staining macrophages, and flow cytometry. The protocol is applicable to both mouse bone-marrow-derived macrophages and human monocyte-derived macrophages. For complete details on the use and execution of this protocol, please refer to Roehle et al. (2021).1 Display omitted •Phagocytosis of live tumor cells by primary mouse or human macrophages•Streamlined protocol for in vitro differentiation of human macrophages from PBMCs•Analysis of phagocytosis in adherent macrophages by flow cytometry•Use of cIAP1/2 antagonism and cytokines to induce phagocytosis Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Although tumor-associated macrophages are generally immunosuppressive, macrophages may also promote tumor clearance via phagocytosis of live tumor cells. Here, we present a protocol for assessing macrophage engulfment of tumor cells in vitro using flow cytometry. We describe steps for cell preparation, reseeding macrophages, and setting up phagocytosis. We then detail procedures for collecting samples, staining macrophages, and flow cytometry. The protocol is applicable to both mouse bone-marrow-derived macrophages and human monocyte-derived macrophages.