Glioblastoma multiform is the most aggressive and invasive brain cancer consisting of genetically and phenotypically altering glial cells. It has massive heterogeneity due to its highly complex and dynamic microenvironment. Here, we used dielectrophoresis to measuree electrophysiological properties of glioma cells and quantified population heterogeneity of U87 glioma cell line. We fabricated a gold microelectrode array within a microfluidic channel and applied 3 Vpp and 100 kHz, 200 kHz, 300 kHz, 400 kHz, 500 kHz, 1 MHz, 2 MHz, 5 MHz, and 10 MHz frequencies to monitor behavior of glioma cells under the microscope. We analyzed dielectrophoretic behavior of 500 glioma cells using the acquired images. We revealed that U87 glioma cells has never experienced negative dielectrophoresis and exhibited crossover frequency around 100 kHz -150 kHz. We detected 3 distinct glioma subpopulations by quantifying dielectrophoretic movement of the cells; 50% of the cells experienced positive dielectrophoresis starting from 500 kHz to 10 MHz, 30% of the cells were spread in the microchannel by moderate DEP forces, and other cells were experienced very weak DEP forces. Our results demonstrate the dielectrophoretic spectra of U87 glioma cell line and associates the dielectrophoretic responses of single glioma cells to the population heterogeneity.