Abstract Adaptations in microdialysis probe designs have made it possible to obtain samples from the extracellular fluid of a variety of tissues with high temporal resolution. The resulting small volume samples, often with low concentration of the analyte(s) of interest, present a particular challenge to the analytical system. Rapid separations can be coupled on-line with microdialysis to provide near real-time data. By combining microdialysis sampling with a liquid chromatographic or capillary electrophoretic separation and a highly sensitive detection method, a separation-based sensor can be developed. Such sensors have been applied to the investigation of drug entities as well as to study endogenous analytes.