Objective: The treatment of glioblastoma (GBM) using low intensity electric fields (~1 V/cm) is being investigated using multiple implanted bioelectrodes, which was termed intratumoral modulation therapy (IMT). Previous IMT studies theoretically optimized treatment parameters to maximize coverage with rotating fields, which required experimental investigation. In this study, we employed computer simulations to generate spatiotemporally dynamic electric fields, designed and purpose-built an IMT device for in vitro experiments, and evaluated the human GBM cellular responses to these fields. &#xD;Approach: After measuring the electrical conductivity of the in vitro culturing medium, we designed experiments to evaluate the efficacy of various spatiotemporally dynamic fields: (a) different rotating field magnitudes, (b) rotating vs. non-rotating fields, (c) 200 kHz vs. 10 kHz stimulation, and (d) constructive vs. destructive interference. A custom printed circuit board (PCB) was fabricated to enable four-electrode IMT in a 24-well plate. Patient-derived GBM cells were treated and analyzed for viability using bioluminescence imaging. &#xD;Main results: The optimal PCB design had electrodes placed 6.3 mm from the center. Spatiotemporally dynamic IMT fields at magnitudes of 1, 1.5, and 2 V/cm reduced GBM cell viability to 58%, 37% and 2% of sham controls respectively. Rotating vs. non-rotating, and 200 kHz vs. 10 kHz fields showed no statistical difference. The rotating configuration yielded a significant reduction (p<0.01) in cell viability (47±4%) compared to the voltage matched (99±2%) and power matched (66±3%) destructive interference cases.&#xD;Significance: We found the most important factors in GBM cell susceptibility to IMT are electric field strength and homogeneity. Spatiotemporally dynamic electric fields have been evaluated in this study, where improvements to electric field coverage with lower power consumption and minimal field cancellations has been demonstrated. The impact of this optimized paradigm on cell susceptibility justifies its future use in preclinical and clinical trial investigations.