This paper reports the characterization and sorting of cells based on stiffness contrast. Cell stiffness is characterized in terms of elastic modulus, deformability index and hydrodynamic resistance. For different cell types, elastic modulus is measured using nanoindentation experiments on AFM and deformability index of cells is measured by hydrodynamic stretching of the cells in a flow focusing microchannel device. Hydrodynamic resistance of cells is obtained by measuring the excess pressure drop across a segment of a microchannel and correlated with cell size ρc and elastic modulus using a large set of experimental data. The highly-invasive malignant breast cancer cells MDA MB 231, non-invasive malignant breast cancer cells MCF 7, human promyelocytic leukaemia cells HL60 and the cervical cancer cells HeLa are considered in the present study. A microfluidic device with focusing and spacing control for stiffness based sorting of cells was designed and fabricated. Experiments were performed to demonstrate cell sorting and characterize the device performance in terms of sorting efficiency, which was found to depend on the stiffness contrast. The proposed device has potential to be used as a lab on chip diagnostic tool for sorting of diseased cells from healthy cells based on stiffness contrast. © The Royal Society of Chemistry.