There is a vast array of dyes currently available for measurement of cytosolic calcium. These encompass single and dual excitation and single and dual emission probes. The choice of particular probe depends on the experimental question and the type of equipment to be used. It is therefore extremely difficult to define a universal approach that will suit all potential investigators. Preparations under investigation are loaded with the selected organic indicator dye by incubation with ester derivatives, by micropipet injection or reverse permeabilization. Indicators can also be targeted to a range of intracellular organelles. Calibration of a fluorescent signal into Ca2+ concentration is in theory relatively simple but the investigator needs to take great care in this process. This chapter describes the theory of these processes and some of the pitfalls users should be aware of. Precise experimental details can be found in the subsequent chapters of this volume.