Drug discovery for CNS disorders is a uniquely challenging endeavor, where calcium (Ca2+) imaging is being actively studied to evaluate the effect of GPCR-targeting drugs in vivo and in vitro. However, the quantification of drug efficacy and targetting corresponding to drug dose and drug type remains challenging. The heterogeneity in the population behavior makes it difficult to decipher the distributions of patterns corresponding to lower and higher drug doses. Herein, we demonstrate the quantification and visualization of Ca2+. imaging data using microstate analysis. This method generalizes the various states of the system at various points in time into fewer defined numbers of microstates appearing in the given data. These microstates and the pattern of their appearance helps to study complex system with a large number of spiking states. Such microstate decomposition can help us to understand and compare the spatiotemporal data. Using this approach, we have attempted to study the heterogeneity in Ca2+ -imaging data obtained from live-cell imaging of rat hippocampal neurons. We have investigated how the nature of microstates changes with respect to drug treatment, size of cell population, and cell sparsity. This analysis can be useful in drug discovery and comparison of the efficacy of different doses of the drug. © 2023 IEEE.