The potential of Raman spectroscopy for ex vivo and in vivo classification of normal and glioblastoma brain tumor development was investigated. High-quality spectra of normal and tumor tissues were obtained using a portable Raman spectrometer coupled to a microprobe with a signal integration time of 5 s. Ex vivo results demonstrated that by using the biochemical information contained in the spectra, we were able to distinguish between normal brain features (white and gray matter), invasion, and tumor tissues with a classification accuracy of 100%. Differences between these features resulted from variations in their lipid signal contributions, which probably reflect differences in the level of myelinization. This finding supports the ability of in vivo Raman spectroscopy to delineate tumor margins during surgery. After implanting C6 cells in rat brain, we monitored, in vivo, the development of glioblastoma tumor from days 0 to 20 post-implantation (PI). The classification exhibited a clear separation of the data into two clusters: one cluster was associated with normal brain tissues (cortex), and the second was related to data measured from tumor evolution. The second cluster could be divided into two subclusters, one associated with tumor tissue from 4 to 13 days PI and the second related to tumor tissue from 15 to 20 days PI. Histological analysis reveals that the differences between these two subclusters are: the presence of a massive infiltration zone in the brain tissue from 4 to 13 days PI, and; a maturation of the tumor characterized by the appearance of edematous and necrotic zones, as well as a diminution in the proliferative and invasive area, from 15 days. This work demonstrates the potential of Raman spectroscopy to provide diagnostic information for the early detection of tumors in vivo.