Phagocytosis is the process by which cells engulf and destroy large particles such as pathogens or apoptotic cells. In this way, macrophages play a pivotal role in the resolution of microbial infections. However, many microorganisms have evolved efficient strategies to preempt the weaponry of macrophages. A better understanding of the components engaged in the phagosome formation and maturation is necessary to devise novel approaches aimed at counteracting these microbial strategies. Recently, large-scale approaches have been used to improve our understanding of phagosome functional properties by the identification of hundreds of proteins and by studying each of them. Presently, purification of pathogen-containing phagosomes presents several technical challenges, whereas the use of latex beads to isolate phagosomes presents many advantages because this system can mimic host-pathogen interactions during phagocytosis. This system thus remains the best approach to advance our knowledge of phagosome biology, notably when used in conjunction with functional approaches. In this chapter, we outline an approach for the isolation of large-scale phagosome preparations with high degrees of purity.