The ultimate goal of phagosomal maturation is the delivery of internalized, particulate cargo to acidic, hydrolytically competent compartments capable of mediating its degradation. Here we outline in detail three fluorometric techniques that allow the study of phagosomal maturation in macrophages by quantifying functionally important features of the lumenal environment of the developing phagosome in real time. The first assay utilizes a particle-restricted, pH-sensitive fluorochrome to measure the acidification of the phagosome. The second reports on the development of the proteolytic capacity of the phagosome by following the hydrolysis of a fluorogenic, generic proteinase substrate. The third quantifies the accumulation of lysosomal constituents within the phagosome by measuring the fluorescence resonance energy transfer (FRET) efficiency between a particle-restricted, donor fluor and a fluid phase acceptor fluor that had been chased previously into lysosomes. The assays are described as population-based methodologies utilizing a spectrofluorometer but, alternatively, can be adapted readily to confocal-based technologies for single phagosomal measurements.