Separation of micron-scale particles and cells is a critical step for a variety of many biological and biomedical studies. Although in the past years, a variety of microfluidic-based techniques have been developed such as Inertial methods, Dielectrophoresis, Magnetophoresis and Acoustophoresis, there is clearly still a demand for a precise, fast, and biocompatible method for separation of microparticles and cells. in the present work, a two-step system has been proposed for particle separation. in the first step particles focused in a serpentine micro channel and in the next step separated by acoustic radiation force. design of geometry is done in SolidWorks and its analysis and simulation are done in three dimensions in Comsol Multiphysics. After studies, two channels (1-circular sinusoidal channel, 2-rectangular zigzag channel) were selected to focus the particles in the first stage, which are well compatible with the acoustic separation region. In this research, no slip boundary condition on walls and bounce condition for collision of the particle to walls are considered. In addition, it is assumed that the collision of the particle to each other is negligible and also particle motion has no effect on fluid flow. First, the laminar flow in the serpentine channel is simulated and the flow field (velocity and pressure) is obtained. Then, the acoustic velocity and pressure field is obtained by the first-order perturbation theory, which is a small perturbation in the Navier-Stokes equations. Finally, it is possible to calculate the force on the particles and obtain their path. In the first system, a sinusial serpentine channel is used to focus the particles. The inlet flow rate from 100 to 400 ?l / min was studied and it was determined that the flow rate of 290 ?l / min is the most suitable mode for pre-focusing the acoustic separation region. Particles with a size of 2 to 15 micrometers were also examined to determine the effect of particles size on their concentration. Finally, it was concluded that particles of 8 and 12 ?m could be focused at a flow rate of 290 ?l / min by a sinusoidal channel, and in the second stage separated by 2 MHz acoustic waves. In the second system, the concentration of 3, 5 and 10 ?m particles in a rectangular zigzag channel in the inlet flow rate of 350 ?l / min was investigated. The results show that the 5 and 10 ?m particles are well concentrated near the side walls of the channel and the 3 ?m particles are less concentrated. In this system, 3 and 10 ?m particles and also 5 and 10 ?m particles can be separated by 1 MHz waves. The results of this study show that by combining hydrodynamics and acoustophoresis, the throughput of the separation system can be significantly increased compared to single-stage methods. Keywords: Particle Separation, Inertial Focusing, Microfluidics, Acoustophoresis, Comsol