Microdialysis is a powerful in vivo technique for the continuous sampling of small molecules within the extracellular fluid space. However, efforts to collect larger molecules have met with little success. To identify biologically active larger molecules in free-moving animals would be of great benefit. For this purpose, we have developed a novel microdialysis method that allows consistent recovery of large molecules from the brain interstitial space in the awake, free-moving mouse. Using a new “vent” probe with a push-pull perfusion system, the present study successfully demonstrated in vivo sampling of pathophysiologically important macromolecules in free-moving mouse brain. This sampling system allowed monitoring of the dynamic changes in their concentrations. Overall, this novel microdialysis system would provide the opportunity to identify the expression patterns of pathophysiologically important proteins in a variety of physiological and pathological processes for a better understanding of various diseases.