Glioblastoma multiforme is one of the most aggressive malignant primary brain tumors. To design effective treatment strategies, we need to better understand the behavior of glioma cells while maintaining their genetic and phenotypic stability. Here, we investigated the deformation and migration profile of U87 Glioma cells under the influence of dielectrophoretic forces. We fabricated a gold microelectrode array within a microfluidic channel and applied sinusoidal wave AC potential at 3 V_pp, ranging from 30 kHz to 10 MHz frequencies, to generate DEP forces. We followed the dielectrophoretic movement and deformation changes of 100 glioma cells at each frequency. We observed that the mean dielectrophoretic displacements of glioma cells were significantly different at varying frequencies with the maximum and minimum traveling distances of 13.22 µm and 1.37 µm, respectively. The dielectrophoretic deformation indexes of U87 glioma cells altered between 0.027–0.040. It was 0.036 in the absence of dielectrophoretic forces. This approach presents a rapid, robust, and sensitive characterization method for quantifying membrane deformation of glioma cells to determine the state of the cells or efficacy of administrated drugs.