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Heterotrimeric GTP-binding proteins (G-proteins) and G-protein-coupled receptors are important signaling components in eukaryotes. In plants, the G-proteins are involved in diverse physiological processes, some of which are exerted via changes in the level of cytosolic free calcium concentration ([Ca
2+
]
cyt
). Various techniques have been developed to measure the change of [Ca
2+
]
cyt
, e.g., calcium-sensitive microelectrodes, chemical fluorescent dyes, and biosensors based on luminescent or fluorescent indicators. In this chapter, we describe a protocol for in vivo [Ca
2+
]
cyt
measurement in G-protein mutants expressing aequorin, a luminescent-based calcium biosensor, to extend our knowledge about G-protein mediated Ca
2+
signaling. This method is also applicable to other early signaling events that are mediated by changes in [Ca
2+
]
cyt
levels.
