High-throughput screening techniques continue to provide important tools to the pharmaceutical industry for the efficient identification of drug leads. However, high-throughput techniques are now being exploited to address a variety of pharmacological and cellular signaling research questions, including the regulation and role of intracellular Ca(2+) in a plethora of biological systems. Although an awareness of specific assay conditions is crucial for reliable and reproducible measurements of intracellular free Ca(2+) whichever system of study is used, the complex temporal nature of Ca(2+) signals has posed some unique limitations for its measurement in high-throughput mode. Progress in high-throughput design has overcome many of these limitations and will complement other technical approaches to understanding the underlying regulation and role of intracellular Ca(2+).