Professional phagocytes function at the hinge of innate and acquired immune responses by internalizing particulate material that is digested and sampled within the phagosome of the cell. Despite intense interest, assays to measure phagosome maturation remain insensitive and few in number. In this current study, we describe three novel assays that quantify important biological properties of the phagosome as it matures. One assay exploits fluorescence resonance energy transfer to quantify mixing of phagocytosed particles carrying a donor fluor with an acceptor fluor loaded previously into the lysosomes as a fluid phase marker. Two additional assays describe the functional maturation of the phagosome as a hydrolytic compartment following the degradation of specifically designed peptide and triglyceride fluorogenic substrates. The peptide substrate is preferentially cleaved by cysteine proteinases, and its degradation reflects proteinase delivery and activation within the acidifying phagosome. The fluorescence emission of the triglyceride analogue profiles the kinetics of triglyceride lipase activity within the phagosome. The fluorescence profiles of all three assays are modulated by known inhibitors of phagosome maturation, demonstrating the veracity, sensitivity and versatility of the assays.