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Abstract Density and mechanical properties (e.g., compressibility or bulk modulus) are important cellular biophysical markers. As such, developing a method to separate cells directly based on these properties can benefit various applications including biological research, diagnosis, prognosis, and therapeutics. As a potential solution, surface acoustic wave (SAW)-based cell separation has demonstrated advantages in terms of biocompatibility and compact device size. However, most SAW-reliant cell separations are achieved using an entangled effect of density, various mechanical properties, and size. In this work, we demonstrate SAW-based separation of cells/particles based on their density and compressibility, irrespective of their sizes, by manipulating the acoustic properties of the fluidic medium. Using our platform, SAW-based separation is achieved by varying the dimensions of the microfluidic channels, the wavelengths of acoustic signals, and the properties of the fluid media. Our method was applied to separate paraformaldehyde-treated and fresh Hela cells based on differences in mechanical properties; a recovery rate of 85% for fixed cells was achieved. It was also applied to separate red blood cells (RBCs) and white blood cells (WBCs) which have different densities. A recovery rate of 80.5% for WBCs was achieved.