High-grade gliomas are highly aggressive central nervous system (CNS) malignancies with high fatality rates if left untreated. There is currently a lack of reliable diagnostic tools to characterize the diffuse cell populations commonly found in these tumors. Here, we report that electrochemical impedance spectroscopy (EIS) can be used in an in vitro system to analyze changes in the impedance contributed by the extracellular matrix (ECM) of two glioblastoma cell lines: GBM 22 and GBM 115. EIS was more effective at resolving differences in impedance from GBM 115 cells than GBM 22 cells, which depended on both cell confluency and frequency. However, differences in impedance were more apparent from the supernatant when the cells were removed in both cell lines. Analysis of the PC12 and either of the GBM cell line co-cultures yielded highly statistically significant differences between all comparisons of cell confluencies and frequency steps. These results illustrate that EIS can be an effective instrument for characterizing the ECM surrounding glioblastoma cells, providing insight into the cellular behavior of these oncogenic cells.