Microdialysis (MD) is a sampling technique that can be employed to monitor biological events both in vivo and in vitro. When it is coupled to an analytical system, microdialysis can provide near real-time information on the time-dependent concentration changes of analytes in the extracellular space or other aqueous environments. Online systems for the analysis of microdialysis samples enable fast, selective and sensitive analysis while preserving the temporal information. Analytical methods employed for online analysis include liquid chromatography (LC), capillary (CE) and microchip electrophoresis and flow-through biosensor devices. This review article provides an overview of microdialysis sampling and online analysis systems with emphasis on in vivo analysis. Factors that affect the frequency of analysis and, hence, the temporal resolution of these systems are also discussed.