In this work, effective cell separation mechanism is proposed in microfluidic device with integration of three inlets out of which one is for suspension of biological cells and another two for effective hydrodynamic focusing of biological cells. The hydrodynamic focusing and its impact on cells as well as channel fluid velocity profile are examined by various designs having different auxiliary inlet angles with 150 μm s−1 auxiliary inlet velocity and 135 μm s−1 main inlet velocity. The yeast cells and the red blood cells (RBCs) modelled as nucleated spherical particles experienced forces in opposite direction with respect to each other due to positive and negative deviation of polarizing properties from the medium leading to separation with good purity. Results have shown good coordination between hydrodynamic focusing and dielectrophoresis resolving the problem associated with the poor separation of biological cells. It has been found that optimizing focusing inlets can give efficient DEP separation and maximizing the separation envelop with lower separation distance leading to miniaturized microfluidic device design with rapid isolation which can further improve the cell viability.