The concentrations of an unbound compound in the blood plasma and brain interstitial fluid provide essential information for studying the permeability of the compound through the blood-brain barrier. Microdialysis is a method for extracting low-molecular-weight substances in body fluids using a semipermeable membrane. By quantifying the concentrations of an unbound compound in dialysate samples obtained from blood microdialysis and intracerebral microdialysis, it is possible to estimate the concentrations of the compound in plasma and in brain interstitial fluid, respectively. A simultaneous blood and brain microdialysis method can facilitate studies on the blood-brain barrier permeability and/or pharmacokinetics/toxicokinetics of a compound in one animal under physiological conditions. This chapter describes the techniques for simultaneous blood and brain microdialysis in a free-moving mouse and examples of their application.