Glioblastoma is a highly invasive and aggressive brain tumor that is very difficult to treat. The rat glioma cell line CNS-1 is a widely used, well-characterized model of infiltrative glioma. We have used CNS-1 to study tumors that initiate within the brain parenchyma. The CNS-1 cells were stably transfected with the yellow fluorescent protein (YFP) variant Venus to enable standard one-photon and two-photon imaging. In this protocol, we describe how to prepare a cranial window and how to inject the tumor cells into the cerebral cortex at a depth suitable for two-photon imaging. Imaging can begin 24 h after implantation of the cells. Two-photon imaging uses a long excitation wavelength (920 nm); the emission spectrum is the same for the fluorophore as for standard one-photon imaging (emission maximum = 528 nm). Two-photon microscopy permits tissue imaging to depths of 500 μm with three-dimensional (3D) high resolution and minimal photodamage to surrounding tissues. Multiple imaging sessions can be conducted over weeks in the same animal, depending on how long the cranial window remains clear and how quickly the tumor grows. Using this technique, the kinetics of tumor growth and invasion into the surrounding brain parenchyma can be measured in the same animal. This model can be used for determining the molecular and cellular players in brain tumor growth and invasion and for testing potential drug therapies to prevent brain metastasis.