Endo-lysosomes are membrane-bound acidic organelles that are involved in endocytosis, recycling, and degradation of extracellular and intracellular material. The membranes of endo-lysosomes express several Ca 2+ -permeable cation ion channels, including two-pore channels (TPC1-3) and transient receptor potential mucolipin channels (TRPML1-3). In this chapter, we will describe four different state-of-the-art Ca 2+ imaging approaches, which are well-suited to investigate the function of endo-lysosomal cation channels. These techniques include (1) global cytosolic Ca 2+ measurements, (2) peri-endo-lysosomal Ca 2+ imaging using genetically encoded Ca 2+ sensors that are directed to the cytosolic endo-lysosomal membrane surface, (3) Ca 2+ imaging of endo-lysosomal cation channels, which are engineered in order to redirect them to the plasma membrane in combination with approaches 1 and 2, and (4) Ca 2+ imaging by directing Ca 2+ indicators to the endo-lysosomal lumen. Moreover, we will review useful small molecules, which can be used as valuable tools for endo-lysosomal Ca 2+ imaging. Rather than providing complete protocols, we will discuss specific methodological issues related to endo-lysosomal Ca 2+ imaging.