Motorized fluorescence microscopy combined with high-throughput microfluidic chips is a powerful method to obtain information about different biological processes in cell biology studies. Generally, to observe different strains under different environments, high-throughput microfluidic chips require complex preparatory work. In this study, we designed a novel and easily operated high-throughput microfluidic system to observe 96 different GFP-tagged yeast strains in one switchable culture condition or 24 different GFP-tagged yeast strains in four parallel switchable culture conditions. A multi-pipette is the only additional equipment required for high-throughput patterning of cells in the chip. Only eight connections are needed to control 96 conditions. Using these devices, the proteomic dynamics of the yeast stress response pathway were carefully studied based on single-cell data. A new method to characterize the proteomic dynamics using a single cell's data is proposed and compared to previous methods, and the new technique should be useful for studying underlying control networks. Our method provides an easy and systematic way to study signaling pathways at the single-cell level.