Glioblastoma (GBM) is a lethal primary brain cancer with a 5.6% five-year survival rate. Tumor treating fields (TTFields) are alternating low-intensity electric fields that have demonstrated a GBM patient survival benefit. We previously reported that 0.5-24 h of TTFields exposure resulted in an increased uptake of FITC-dextran fluorescent probes (4-20 kDa) in human GBM cells. However, this approach, in which a fluorescence plate-based detector is used to evaluate cells attached to glass coverslips, cannot distinguish FITC-dextran uptake in live vs. dead cells. The goal of the study was to report the optimization and validation of two independent methods to quantify human GBM cell membrane permeabilization induced by TTFields exposure. First, we optimized flow cytometry by measuring mean fluorescence intensity at 72 h for 4 kDa (TTFields 6726 ± 958.0 vs. no-TTFields 5093 ± 239.7, p = 0.016) and 20 kDa (7087 ± 1137 vs. 5055 ± 897.8, p = 0.031) probes. Second, we measured the ratio of lactate dehydrogenase (LDH) to cell viability (measured using the CellTiter-Glo [CTG] viability assay); the LDH/CTG ratio was higher under TTFields (1.47 ± 0.15) than no-TTFields (1.08 ± 0.08) conditions, p < 0.0001. The findings using these two independent methods reproducibly demonstrated their utility for time-dependent evaluations. We also showed that these methods can be used to relate the cell membrane-permeabilizing effects of the non-ionizing radiation of TTFields to that of an established cell membrane permeabilizer, the non-ionic detergent Triton-X-100. Evaluating carboplatin ± TTFields, the LDH/CTG ratio was significantly higher in the TTFields vs. no-TTFields condition at each carboplatin concentration (0-30 µM), p = 0.014. We successfully optimized and validated two cost-effective methods to reproducibly quantify TTFields-induced human GBM cancer cell membrane permeabilization.