Dielectric spectroscopy is a label-free, non-contact, real-time, multi-layer sensing technology, and has been used for identification and quantification of many biological materials. A combination of such sensing features is in demand for monitoring of organ-on-chip systems; however available sensing technologies have yet to address this need. In this work, we explore the possibility of leveraging the inherent features of dielectric spectroscopy for the application in organ-on-chip systems, by investigating three key technological developments using open-ended coaxial probes. Firstly, biocompatible non-contact sensing capabilities are proved by showing similar sensing performance of Parylene C-coated probes and uncoated probes. Secondly, a setup and methodology are developed for highly accurate and non-destructive height positioning of the probe to allow for precise extraction of intermediate sample layers. Finally, non-contact multi-layer sensing performance of the presented technology is successfully demonstrated by means of a biological phantom in a three-layered system. With further integration, dielectric spectroscopy can potentially become a cornerstone sensing technique for organ-on-chip by enabling real-time non-contact tracking of various tissue contents and properties.