Migration of cancer cells from the primary organ site via the bloodstream to distant sites is critical to the development of malignant metastasis and is in part determined by soluble host factors in the serum. Conventional Boyden chamber assays to evaluate cell motility require high volumes of reagents and are impractical for high-throughput analysis. We have designed and evaluated a poly-dimethylsiloxane (PDMS) microfluidic device in order to systematically study cancer cell migration. Photolithography and soft lithography processes were used to fabricate the PDMS devices from a negative photoresist (SU-8) mold. The device provides two separate identical chambers that are interconnected by an array of identical narrow channels, high, wide, and long. One chamber is seeded with cancer cells whose migration characteristics are to be evaluated, while the other chamber contains media with chemoattractants toward which the cancer cells migrate. In this microfluidic chamber model, the migration of cancer cells within and across the microfluidic channels over a prescribed time was quantified using time-lapse photographs. The microfluidic chamber is a cost-effective platform that uses small volumes of reagents, can maintain stable chemokine gradients, allow real-time quantitative study of cancer cell migration, and provide information about cellular dynamics and biomechanical analysis. This work demonstrated the utility of the microfluidic device as a platform to study cancer cell migration as well as the potential applications in the identification of specific chemokine agents and development of drugs targeting cell migration.