This paper proposes a novel concept of dielectrophoresis (DEP)-active hydrophoretic focusing of micro-particles and murine erythroleukemia (MEL) cells. The DEP-active hydrophoretic platform consists of crescent shaped grooves and interdigitated electrodes that generate lateral pressure gradients. These embedded electrodes exert a negative DEP force onto the particles by pushing them into a narrow space in the channel where the particle to groove interaction is intensive and hydrophoretic ordering occurs. Particles passing through the microfluidic device are directed towards the sidewalls of the channel. The critical limitation of DEP operating at a low flow rate and the specific hydrophoretic device for focusing particles of given sizes were overcome with the proposed microfluidic device. The focusing pattern can be modulated by varying the voltage. High throughput was achieved (maximum flow rate ~150 μL min−1) with good focusing performance. The non-spherical MEL cells were utilised to verify the effectiveness of the DEP-active hydrophoretic device.