Recently, surface acoustic wave (SAW) based acoustofluidic separation of microparticles and cells has attracted increasing interest due to accuracy and biocompatibility. Precise control of the input power of acoustofluidic devices is essential for generating optimum acoustic radiation force to manipulate microparticles given their various parameters including size, density, compressibility, and moving velocity. In this work, an acoustophoretic system is developed by employing SAW based interdigital electrode devices. Power meters are applied to closely monitor the incident and reflected powers of the SAW device, which are associated with the separation efficiency. There exists a range of input powers to migrate the microparticles to the pressure node due to their random locations when entering the SAW field. Theoretical analysis is performed to predict a proper input power to separate mixtures of polystyrene microspheres, and the end lateral position of microspheres being acoustically separated. The separation efficiency of four sizes of microspheres, including 20 µm, 15 µm, 10 µm, and 5 µm, is calculated and compared with experimental results, which suggest the input power for separating the mixture of these microspheres. The study provides a practical guidance on operating SAW devices for bioparticle separation using the incident power as a control parameter.