Cells as an intricate organism can orchestrate biochemical signals and initiate complex physiological responses when they are exposed to external stimuli including mechanical,chemical and electrical stimulations.[1] As one type of chemical stimulations,hypertonicity always arouse several cellular responses,such as cell shrinkage (volume decrease) and the underlying ion movement (K+,Na+,Cl-,Ca2+),which have been regarded as a hallmark of the programmed cell death by apoptosis.[2] To study the calcium cellular response to the hypertonic condition,cells are exposed to the hypertonic solution without concentration gradients,by which it is hard to study the dynamics of cell responses during the increasing cell shrinkage.Moreover,in order to study single cell behaviors in the hypertonic condition,patch clamp is used to immobilize single cells,however,it is rather labor-intensive,time-consuming and inherently incompatible with the high-throughput analysis.To solve these problems,here we demonstrated an evaporation-assisted method that allows for the formation of a hypertonic solution with dynamically changing concentration for the study of calcium response at single cell level in high-throughput.Five different kinds of cells (HL60,Jurkat,A549,H460,LCSCs) were investigated and we found the abrupt calcium increase when cells exposed to the air at the end of evaporation.Also,the cellular calcium response showed both obvious cell-type-specific differences and single-cell heterogeneity.Especially,compared to A549 and H460,lung cancer stem cells showed the maximum response and the widest response distribution which indicated the essential role of Ca2+.These results demonstrate that this evaporation-assisted method provides a useful approach for investigating the intracellular response to increasing stimuli in high-throughput.