Plants use different signaling pathways to respond to various external stimuli. The adaptation of plants to frequent environmental changes requires their ability to sense and to respond rapidly to these stimuli. Plants use intracellular/cytosolic Ca2+ to react to the changing environment. In the course of evolution, Ca2+ has emerged as a versatile intracellular messenger carrying information to most cellular processes that are important for cell life. Ca2+ as a second messenger initiates many responses involved in growth, development, symbiosis and defense. Most environmental challenges faced by plants including biotic and abiotic stress have been shown to induce changes in the cytosolic Ca2+ ([Ca2+]cyt). Biotic agents or stress molecules binding to its putative plasma membrane receptors provoke a large transient and sustained [Ca2+]cyt influx which triggers numerous downstream events leading to many adaptation and developmental processes. Intracellular release of Ca2+ is one of the earliest events in signal perception and it plays major roles in almost all aspects of plant’s life. As [Ca2+]cyt is involved in various signaling and developmental processes, increasing interest has been focussed on the measurement of [Ca2+]cyt in the cell/tissue/organ/organism without destructing the cells/tissues. Here we discuss different methods used to measure [Ca2+]cyt qualitatively and/or quantitatively with their advantages and disadvantages. As our laboratory is using aequorin technology to measure [Ca2+]cyt in the model system involving Arabidopsis thaliana and a plant growth promoting endophytic fungus, Piriformospora indica, more emphasis is given on this technique.