Professional phagocytes ingest particulate material to fulfil a diverse array of functions in a multicellular organism. The ancestral function of phagosomes is digestion; however, through evolution this degradative capacity has become pivotal to the adaptive immune response by processing antigens to be presented to lymphocytes. Moreover, phagocytes have also acquired an active role in microbial killing. This Innovation article describes new assays that probe the biological activities which occur within phagosomes. These assays provide functional insights into how the phagosome fulfils its diverse roles in homeostasis and in innate and adaptive immune responses.