In this work, we describe the separation of embryonic mouse hippocampal neurons from glial cells using a positive dielectrophoresis (DEP) process. Here, we have implemented a cell trapping-favorable, cell suspension solution with low conductivity. It enables positive dielectrophoresis for hippocampal neurons (thereby attracting them to the electrodes), while resulting in negative dielectrophoresis for glial cells (repelling them from the electrodes). We have systematically performed a mathematical simulation and analysis to anticipate the DEP frequency at which hippocampal neurons and glial cells are separated. Simulated DEP crossover frequencies have been experimentally verified, and new, refined glial dielectric and physical properties are suggested that better reflect the experimental results obtained. DEP movements of neurons and glial cells in targeted separation media were experimentally analyzed, under the specified electric signal. Additionally, we have confirmed our modeling results by selectively trapping neurons over electrodes on a custom-made, multi-electrode array (MEA), resulting in active recruitment of neurons over the stimulation and recording sites. This technique is a valuable addition to the toolbox for creating more functional and versatile multi-electrode arrays.