Polymeric separators have been developed since 2010 to produce acoustophoretic separation of particles or cells in suspension with high efficiency. They rely on three-dimensional (3D) resonances of their whole structure actuated by ultrasounds. In this paper, a numerical 3D analysis is presented and validated as the only tool for optimization of these polymeric chips to perform efficient separation applications. In contrast to conventional acoustophoretic techniques based on the establishment of standing waves in the liquid phase of the channel (requiring rigid chip materials, such as silicon or glass), whole-structure resonances of the chip allow the use of materials that are acoustically soft and of low acoustic impedance, which is close to that of the liquid samples hosted. The resonance requirement is not restricted to the liquid phase in the polymeric chips, but it extends to the 3D whole structure, allowing any material. It provides significant advantages in the design and manufacture of our chips, allowing the use of low-cost materials and cheap manufacturing processes and even printing of devices. The extraordinary complexity of their multiple resonances requires theoretical approaches to optimize their acoustophoretic performance. Hence, the importance of 3D numerical analyses, which are capable of predicting the acoustic behavior of these chips, is to perform acoustophretica separation in suspensions.