The efficient degradation of internalized particulate matter is a principal objective of the macrophage’s phagosome. Assessment of the true hydrolytic capacity within the phagosomal lumen is often difficult as it is subject to many factors beyond recruitment of lysosomal hydrolases. Here we outline three assays that allow quantitative measurements of serine-cysteine protease, triglyceride lipase, and β-galactosidase activities within the phagosomes of macrophages, in real time. The assays utilize ratio fluorometry between particle-associated fluorogenic substrates and calibration fluorochromes to yield internally controlled values that record rates of substrate hydrolysis. The methods described utilize a spectrofluorometer for fluorometric measurements from a population of macrophages. These assays, however, can be expanded to high-throughput or single cell formats. In addition, this approach can be applied to measure a wide variety of phagosomal hydrolytic properties with the design of suitable fluorogenic substrates.