Interferon-gamma IFN-γ)-induced remodeling of the bacterial phagosome for pathogen clearance elicits the aid of a new family of GTPases termed the p47 IRGs. Members of this group reside primarily on ER-Golgi membranes before translocating to the nascent phagosome within minutes of bacterial uptake. Recruitment of p47 IRGs coincides with the acquisition of phagosome maturation and autophagy markers as well as enhanced acidification of this organelle. Here we describe a simple spectrofluorometric assay to measure luminal acidification of the bacterial phagosome within intact cells such as macrophages. This method can be applied to study the phagosomal pH pH-pg) of activated cells infected with a variety of infectious microorganisms and the roles played by members of the p47 IRG family in auto)phagolysosome biogenesis.