Calcium (Ca(2+)) has long been recognized as a crucial intracellular messenger attaining stimuli-specific cellular outcomes via localized signaling. Ca(2+)-binding proteins, such as calmodulin (CaM), and its target proteins are key to the segregation and refinement of these Ca(2+)-dependent signaling events. This review not only summarizes the recent technological advances enabling the study of subcellular Ca(2+)-CaM and Ca(2+)-CaM-dependent protein kinase (CaMKII) signaling events but also highlights the outstanding challenges in the field.