Fluorescence microscopy and image analysis are powerful techniques to examine the distribution and interactions of different cellular compartments, including mammalian organelles with intravacuolar pathogens. Toxoplasma gondii is an obligate intracellular protozoan parasite that forms a membrane-bound compartment, the parasitophorous vacuole (PV), upon invasion of mammalian cells. From within the PV, the parasite interacts with many host organelles (without fusion), redirects host vesicles decorated with Rab GTPases to the PV, and internalizes many of these nutrient-filled Rab vesicles into the PV. Here, we report a method to distinguish the host Rab vesicles that are exclusively trapped in the Toxoplasma PV from those localized along the edge of the vacuole. Such a discrimination between the two Rab vesicle populations (inside versus outside of the PV) allows the selective characterization of the intra-PV Rab vesicles, for example, number per PV, volume, and distance from the PV centroid, as well as comparisons between wild-type and mutant Toxoplasma.