In this study, we demonstrate the use of cyclical low frequency signals with insulator-based dielectrophoresis (iDEP) devices for the separation of particles of similar characteristics and an experimental method for estimating particle DEP mobilities. A custom signal designer program was created using Matlab® and COMSOL Multiphysics® for the identification of specific low frequency signals aimed at separating particle mixtures by exploiting slight differences in surface charge (particle zeta potential) or particle size. For the separation by surface charge, a mixture of two types of 10 μm particles was analyzed and effectively separated employing both a custom step signal and a sawtooth left signal. Notably, these particles had the same shape, size, and surface functionalization as well as were made from the same substrate material. For the separation by size, a sample containing 2 μm and 5 μm particles was successfully separated using a custom step signal; these particles had the same shape, surface functionalization, were made from the same substrate materials, and had only a small difference in zeta potential (10 mV). Additionally, an experimental technique was developed to estimate the dielectrophoretic mobility of each particle type; this information was then utilized by the signal designer program. The technique developed in this study is readily applicable for designing signals capable of separating micron-sized particles of similar characteristics, such as microorganisms, where slight differences in cell size and the shape of surface charge could be effectively exploited. These findings open the possibility for applications in microbial screening using iDEP devices.