Measurement of cytosolic calcium activity in single neuron and quantification of response to drug treatment over a longer period remain challenging. Especially, analysis of amplitude, frequency, and delay in calcium response in G-protein coupled receptors (GPCR) targeting drug-treated neurons is complicated due to inherent heterogeneity. To address this, we have utilized k-means clustering to identify various sub-populations. Here we focus on measuring drug dose response in the single neuron by means of high-resolution confocal imaging. Metabotropic glutamate receptor was activated using 3,5-dihydroxyphenylglycine (DHPG). The result reveals an unusual delay in calcium response for higher dose particularly in responding neurons. The proposed methodology can be used for selection of drug dose and optimization of the time window for neurodegenerative diseases. © 2019 IEEE.