This letter presents a biosensor first developed to perform microwave dielectric spectroscopy (MDS) analysis, which is evaluated for in situ electroporation (EP) at the single- cell level. After demonstrating the possibility to characterize living cancer cells in the radio-frequency (RF) sensor by acquir- ing their dielectric properties, we then evaluate the use of this RF sensor to apply high electric field stimulus to trigger cell permeabilization. The impact of applied voltage and therefore electric field intensity on the number of permeabilized cells is defined, leading to the establishment of an EP threshold, which is EP conditions and cell dependent. This paves the way for further studies assessing the impact of in situ EP using the MDS method performed with a unique microdevice and consequent observation and understanding of the EP phenomenon.