We report a novel approach to quantify interstitial analytes in living tissue by combining open-flow microperfusion (OFM) with a sensor and the re-circulation method. OFM is based on the unrestricted exchange of molecules between the interstitial fluid (ISF) and a perfusion medium through macroscopic perforations that enables direct access to the ISF. By re-circulating the perfusate and monitoring the changes of the analytes' concentration with a sensor, the absolute analyte concentration in the ISF can be calculated. In order to validate the new concept, the absolute electrical conductivity of the ISF was identified in six subjects to be 1.33 ± 0.08 S/m (coefficient of variation CV = 6 %), showing the robustness of this approach. The most striking feature of this procedure is the possibility to monitor several compounds simultaneously by applying different sensors which will allow not only the determination of the concentration of a single substance in vivo but also the simultaneous dynamics of different analytes. This will open new fields in analytical chemistry, pharmacology, as well as clinical experimental research.