The mechanical properties of a cell, which include parameters such as elasticity, inner pressure, and tensile strength, are extremely important because changes in these properties are indicative of diseases ranging from diabetes to malignant transformation. Considering the heterogeneity within a population of cancer cells, a robust measurement system at the single cell level is required for research and in clinical purposes. In this study, a potential microfluidic device for high-throughput and practical mechanotyping were developed to investigate the deformability and sizes of cells through a single run. This mechanotyping device consisted of two different sizes of consecutive constrictions in a microchannel and measured the size of cells and related deformability during transit. Cell deformability was evaluated based on the transit and on the effects of cytoskeleton-affecting drugs, which were detected within 50 ms. The mechanotyping device was able to also measure a cell cycle without the use of fluorescent or protein tags.