Imaging cytosolic calcium in neurons is emerging as a new tool in neurological disease diagnosis, drug screening, and toxicity testing. Ca 2+ oscillation signatures show a significant variation depending on GPCR targeting agonists. Quantification of Ca 2+ spike trains in ligand induced Ca 2+ oscillations remains challenging due to their inherent heterogeneity in primary culture. Moreover, there is no framework available for identification of optimal number of clusters and distance metric to cluster Ca 2+ spike trains. Using quantitative confocal imaging and clustering analysis, we show the characterization of Ca 2+ spiking in GPCR targeting drug-treated primary culture of hippocampal neurons. A systematic framework for selection of the clustering method instead of an intuition-based method was used to optimize the cluster number and distance metric. The results discern neurons with diverse Ca 2+ response patterns, including higher amplitude fast spiking and lower spiking responses, and their relative percentage in a neuron population in absence and presence of GPCR-targeted drugs. The proposed framework was employed to show that the clustering pattern of Ca 2+ spiking can be controlled using GABA B and mGluR targeting drugs. This approach can be used for unbiased measurement of neural activity and identification of spiking population with varying amplitude and frequencies, providing a platform for high-content drug screening. © 2018 American Chemical Society.