The purpose of this study is to demonstrate particle separation through a novel mechanism termed as “time series alternate flow” using a microdevice as it is a real challenge to separate particles with a narrow size range (i.e., or smaller), especially achieving particles separation through the hydrodynamic method without the help from additional flow or force fields. High fidelity computational fluid dynamics with particle trajectory approach was employed for simulations. Particle separation of different sizes in the range size was achieved by operating the microdevice at various actuating frequencies. The results obtained indicated that the proposed mechanism is feasible for particle separation of multiple sizes. Our novel mechanism proposed potentially represents a viable microtechnological approach for particle separation in many drug delivery applications.