Genetically encoded Ca²⁺ indicators allow researchers to quantitatively measure Ca²⁺ dynamics in a variety of experimental systems. This protocol summarizes the indicators that are available, and highlights those that are most appropriate for a number of experimental conditions, such as measuring Ca²⁺ in specific organelles and localizations in mammalian tissue-culture cells. The protocol itself focuses on the use of a cameleon, which is a fluorescence resonance-energy transfer (FRET)-based indicator comprising two fluorescent proteins and two Ca²⁺-responsive elements (a variant of calmodulin (CaM) and a CaM-binding peptide). This protocol details how to set up and conduct a Ca²⁺-imaging experiment, accomplish offline data processing (such as background correction) and convert the observed FRET ratio changes to Ca²⁺ concentrations. Additionally, we highlight some of the challenges in observing organellar Ca²⁺ and the alternative strategies researchers can employ for effectively calibrating the genetically encoded Ca²⁺ indicators in these locations. Setting up and conducting an initial calibration of the microscope system is estimated to take ∼1 week, assuming that all the component parts are readily available. Cell culture and transfection is estimated to take ∼3 d (from the time of plating cells on imaging dishes). An experiment and calibration will probably take a few hours. Finally, the offline data workup can take ∼1 d depending on the extent of analysis.