The goal of this study was to investigate the in vivo extracellular kinetics of doxorubicin (Dox) in glioblastoma multiforme (GBM)-bearing mice following focused ultrasound (FUS)-induced blood-brain barrier (BBB) disruption using microdialysis. An intracranial brain tumor model in NOD-scid mice using human brain GBM 8401 cells was used in this study. Prior to each sonication, simultaneous intravenous administration of Dox and microbubbles, and the Dox concentration in the brains was quantified by high performance liquid chromatography (HPLC). Drug administration with sonication elevated the tumor-to-normal brain Dox ratio of the target tumors by about 2.35-fold compared with the control tumors. The mean peak concentration of Dox in the sonicated GBM dialysate was 10 times greater than without sonication, and the area under the concentration-time curve was 3.3 times greater. This study demonstrates that intracerebral microdialysis is an effective means of evaluating real-time target BBB transport profiles and offers the possibility of investigating the pharmacokinetics of drug delivery in the sonicated brain.