To advance existing in vitro cell impedance spectroscopy and improve reliability of impedimetric sensors for cell analysis, we have electrically insulated interdigitated microelectrodes using the high-k biomaterial zirconium dioxide (ZrO₂). We report the smallest passivation thickness to electrode distance ratio of 10⁻³ using atomic layer deposition resulting in electrically insulated metal oxide films of 15 nm thickness. For the first time the influence of the insulation on sensor performance is experimentally and theoretically analyzed using numerical simulations. In addition an equivalent electrical circuit model was established and validated using non-linear least square fitting. Results of the computational simulations revealed improved electrical current distribution across the electrically insulated interdigitated electrode structures in comparison to open (not passivated) electrodes. Furthermore, we found linear decrease of current density in z-direction within 5 μm distance from the sensor surface in the presence of ZrO₂ nanocoatings is ideally suited to assess confluent cell layers. Final practical application of the ZrO₂ nanolayer passivated impedimetric sensors is demonstrated for nanotoxicological investigations, where sensitivity and repeatability are crucial parameters for cell analysis. Results of our study show that the reproducible and standardizable deposition of a uniform metal oxide nanocoating improves current density distributions, has no performance drawbacks compared to open sensors and enables sensitive detection of protein-coating effects on cytotoxic silica nanoparticles. The presented novel sensor design allows for the integration of alternative electrode materials such as aluminum enabling cost-effective fabrication of large-volume sensor arrays.