The lysosome is the main catabolic organelle in the cell, also serving as a signaling platform. Lysosomes maintain a low intraluminal pH where dozens of hydrolytic enzymes degrade a wide variety of macromolecules. Besides degradation of polymers, the lysosome is involved in various cellular processes, including energy metabolism, plasma membrane repair and antigen presentation. Recent work has shown that the lysosome is an important calcium store, modulating diverse cellular functions such as membrane fusion and fission, autophagy and lysosomal biogenesis. Precise measurement of free lysosomal calcium concentration has been hampered by its low luminal pH, since the affinity of most calcium probes decreases with higher proton concentration. Here we detailed an adapted protocol for the simultaneous measurement of lysosomal pH and calcium using dextran-conjugated ratiometric fluorescent dyes. As compared with indirect measurements of lysosomal calcium release using genetically-encoded calcium indicators (GECIs), the present method offers the possibility of obtaining pH-corrected, intraluminal calcium concentrations at single lysosome resolution. It also enables simultaneous temporal resolution of lysosomal calcium and pH.