Cellular components sequestered by autophagosomes during macroautophagy must be degraded and their components recycled in order to maintain homeostasis. To this end cells orchestrate the fusion of autophagosomes with lysosomes, degradative organelles that are rich in hydrolases. Most of the lysosomal enzymes function optimally at low pH, and products of macromolecular catabolism are cotransported with protons across the autolysosomal membrane. These functions are facilitated by the ability of lysosomes to pump protons inward, acidifying their lumen. Clearly, proper homeostasis of the luminal pH is crucial for autolysosomal function. We describe a method for the measurement of the absolute pH of individual autolysosomes in live cells. This technique involves measurement of the fluorescence of a pH-sensitive probe initially delivered to lysosomes and subsequently determined to have reached autolysosomes. By measuring the fluorescence at two separate wavelengths and calculating their ratio, potential artifacts introduced by photobleaching or by changes in autolysosome size, shape, or positioning are minimized. Combining such ratio determinations with an in situ calibration procedure enables absolute measurements of pH, which are superior to the qualitative estimates obtained with fluorescent weak bases such as LysoTracker.