Due to the constant perfusion of the microdialysis probe with fresh perfusate, a total equilibrium cannot be established. This results in dialysate concentrations that are lower than those measured at the distant sampling site. In order to correlate concentrations measured in the dialysate with those present at the distant sampling site, a calibration factor (recovery) is needed. The recovery can be determined at steady-state using the constant rate of analyte exchange across the microdialysis membrane.  The rate at which an analyte is exchanged across the semipermeable membrane is generally expressed as the analyte’s extraction efficiency.  The extraction efficiency is defined as the ratio between the loss/gain of analyte during its passage through the probe (C−C) and the difference in concentration between perfusate and distant sampling site (C−C).
In theory, the extraction efficiency of a microdialysis probe can be determined by: 1) changing the drug concentrations while keeping the flow rate constant or 2) changing the flow rate while keeping the respective drug concentrations constant.  At steady-state, the same extraction efficiency value is obtained, no matter if the analyte is enriched or depleted in the perfusate.  Microdialysis probes can consequently be calibrated by either measuring the loss of analyte using drug-containing perfusate or the gain of analyte using drug-containing sample solutions. To date, the most frequently used calibration methods are the low-flow-rate method, the no-net-flux method, the dynamic (extended) no-net-flux method, and the retrodialysis method.  The proper selection of an appropriate calibration method is critically important for the success of a microdialysis experiment.  Supportive in vitro experiments prior to the use in animals or humans are therefore recommended.  In addition, the recovery determined in vitro may differ from the recovery in humans.  Its actual value therefore needs to be determined in every in vivo experiment.