Focusing and separation of microparticles in a complex mixture have had wide applications in chemistry, biology, medicine, etc. This work presents a numerical and experimental investigation on focusing and continuous separation of microparticles in a geometrically optimized arrangement of steps in the form of a staircase using insulator-based dielectrophoresis (iDEP) mechanism. First, a detailed finite element analysis was performed on important parameters in the focusing and separation of microparticles, such as geometry of stair-shaped microchannel, total voltage, and voltage difference applied to reservoirs. The optimum parameters obtained from numerical analysis were used for experimental work. Theoretically, predicted microparticle trajectories are in good agreement with experimentally observed ones. Experimental and numerical results show that the performance of focusing of microparticles enhances with growth of the total voltage (in a constant voltage difference) and decreases with voltage difference. The fabricated iDEP microchip enhances the performance of focusing and separation of microparticles due to its stair-shaped microchannel and therefore operates at low DC total applied voltages of 90-110 V.