The behavior of microglial cells involves the activity of a variety of ion channels and ion transporters, which are implicated in the regulation of ion concentrations, membrane potential, and cell volume of microglia. Fluorescence imaging has been proven to be an elegant method to study ion concentration changes in intact microglial cells under physiological and pathophysiological conditions. The development of highly specific ion indicators has made it possible to detect changes in intracellular Ca(2+), Na(+), and H(+) concentrations of microglial cells as a result of ion channel or ion transporter activity. Fluorescence signals of isolated dye-loaded microglial cells can be detected via a CCD camera equipped to a conventional microscope. This chapter summarizes protocols of loading of microglial cells with small-molecule ion indicators as well as protocols optimal for measurement and analysis of intracellular Ca(2+), Na(+), and H(+) concentrations in microglia in vitro.