Calcium dynamics is crucial for many signaling pathways and cell functions. Understanding how calcium regulates cell function often requires long-term imaging of calcium dynamics. Here we report a methodological approach of long-term (5-10 h) imaging of calcium dynamics in cultured cells. The approach links calcium imaging using genetically encoded calcium indicators and semi-automatic tracking of individual cells. It can be used in a large variety of situations, ranging from the role of calcium in biological processes to cell heterogeneity and screening of drugs modifying signaling pathways.