Microfluidic platforms have been widely considered as an enabling technology for studying the ion transport phenomena of cells under precisely controlled shear stresses. Here, we report the application of a unique microfluidic platform to analyze the response of transgenic TRPV4-HEK293 cells in response to different shear stresses and in one field of view. Applying this system, we show the kinetics of calcium signalling at different shear stresses in TRPV4 positive cells and elucidate the threshold of their response. We show that there is direct correlation between the magnitude of shear stress and percentage of cells that are able to sense that level of shear. Further, we show that shear stress-induced elevation in intracellular calcium levels ([Ca2+]i) is through calcium influx from extracellular sources. The results demonstrate that the microfluidic system has unique capabilities for analysis of shear stress on adhesive cells and that it should be amenable to moderate throughput applications.