During calibration with the no-net-flux-method, the microdialysis probe is perfused with at least four different concentrations of the analyte of interest (C) and steady-state concentrations of the analyte leaving the probe are measured in the dialysate (C). The recovery for this method can be determined by plotting C−C over C and computing the slope of the regression line. If analyte concentrations in the perfusate are equal to concentrations at the sampling site, no-net flux occurs. Respective concentrations at the no-net-flux point are represented by the x-intercept of the regression line. The strength of this method is that, at steady-state, no assumptions about the behaviour of the compound in the vicinity of the probe have to be made, since equilibrium exists at a specific time and place. However, under transient conditions (e.g. after drug challenge), the probe recovery may be altered resulting in biased estimates of the concentrations at the sampling site. To overcome this limitation, several approaches have been developed that are also applicable under non-steady-state conditions. One of these approaches is the dynamic no-net-flux method.