The delivery of intravenously administered cancer therapeutics to brain tumors is limited by the blood-brain barrier. A method to directly image the accumulation and distribution of macromolecules in brain tumors in vivo would greatly enhance our ability to understand and optimize drug delivery in preclinical models. This protocol describes a method for real-time in vivo tracking of intravenously administered fluorescent-labeled nanoparticles with two-photon intravital microscopy (2P-IVM) in a mouse model of glioblastoma (GBM). The protocol contains a multi-step description of the procedure, including anesthesia and analgesia of experimental animals, creating a cranial window, GBM cell implantation, placing a head bar, conducting 2P-IVM studies, and post-surgical care for long-term follow-up studies. We show representative 2P-IVM imaging sessions and image analysis, examine the advantages and disadvantages of this technology, and discuss potential applications. This method can be easily modified and adapted for different research questions in the field of in vivo preclinical brain imaging.