The aim of this study was to assess feasibility and accuracy of a hand-held, intraoperative Raman spectroscopy device as a neuronavigation aid to accurately detect neoplastic tissue from adjacent normal gray and white matter. Although Raman spectra are complicated fingerprints of cell signature, the relative shift corresponding to lipid and protein content (2,845 and 2,930 cm−1, respectively), can provide a rapid assessment of whether tissue is normal white or gray matter vs. neoplasia for real-time guidance of tumor resection. Thirteen client-owned dogs were initially enrolled in the study. Two were excluded from final analysis due to incomplete data acquisition or lack of neoplastic disease. The diagnoses of the remaining 11 dogs included six meningiomas, two histiocytic sarcomas, and three gliomas. Intraoperatively, interrogated tissues included normal gray and/or white matter and tumor. A total of five Raman spectra readings were recorded from the interrogated tissues, and samples were submitted for confirmation of Raman spectra by histopathology. A resultant total of 24 samples, 13 from neoplastic tissue and 11 from normal gray or white matter, were used to calculate sensitivity and specificity of Raman spectra compared to histopathology. The handheld Raman spectroscopy device had sensitivity of 85.7% and specificity of 90% with a positive predictive value of 92.3% and negative predictive value of 81.6%. The Raman device was feasible to use intraoperatively with rapid interpretation of spectra. Raman spectroscopy may be useful for intraoperative guidance of tumor resection.