Increased deformability and softness endow tumor cells with highly invasive and metastatic capabilities. We exploited these characteristics to fabricate a high-throughput microfluidic device to measure cell deformability and separate cancer cells. Driven by hydrodynamic forces, the cells with better deformability passed through the chip faster, whereas stiffer cells passed through the device over a longer time period. The MDA-MB-231 and MCF-7 cell lines were used to evaluate the device because their metastatic potentials were known. We found that MDA-MB-231 cells, which were softer and exhibited stronger deformability, passed through the device more quickly. HeLa cells were also successfully separated into softer and stiffer subpopulations, whose distinct mechanical properties were confirmed by atomic force microscopy. We also measured the expression of metastasis-associated proteins (epidermal growth factor receptor and integrin β 1) and found that subpopulations with varied deformabilities had different expression levels. Our results suggested that this high-throughput microfluidic device could be used to screen and evaluate the curative effects of drug and cancer progression by simultaneously testing cell deformability and expression levels of metastasis-associated proteins in separated cell subpopulations.