We have developed a single-cell assay platform that allows quantitative analysis of single cell chemotaxis by dynamic morphogenetic gradients, subcellular microscopic imaging and automated image analysis, and have applied these to measure cellular polarization of budding yeast. The computer-controlled microfluidic device regulates the gradient profile at any given time, and allows quantitative monitoring of cell morphology and the localization and expression of specific marker proteins during the dynamic polarization process. With this integrated experimental system, we compare the polarized signaling response of wild-type and far1-H7 mutant cells, which express a truncated Far1 protein unable to interact with Cdc24. Our results confirm that Far1 functions as an adaptor that recruits polarity establishment proteins to the site of extracellular signaling. Moreover, by changing the gradient profile and estimating the number of bound surface receptors, we quantitatively address why surprisingly small differences in pheromone concentration across yeast cells can be amplified into a robust polarity axis. This integrated single cell experimental platform thus opens the possibility to quantitatively investigate the molecular regulatory mechanism of chemotaxis in yeast, which serves as a paradigm to understand the fundamental processes involved in cancer metastasis, angiogenesis and axon generation.