Tumor-positive resection margins are a major problem in oral cancer surgery. High-wavenumber Raman spectroscopy is a reliable technique to determine the water content of tissues which may contribute to differentiate between tumor and healthy tissue. The aim of this study was to examine the use of Raman spectroscopy to differentiate tumor from surrounding healthy tissue in oral squamous cell carcinoma. From fourteen patients undergoing tongue resection for squamous cell carcinoma, the water content was determined at 170 locations on freshly excised tongue specimens using the Raman-bands of the OH-stretching vibrations (3350-3550cm-1) and of the CH-stretching vibrations (2910-2965cm-1). The results were correlated with histopathological assessment of hematoxylin and eosin stained thin tissue sections obtained from the Raman measurement locations. The water content values from squamous cell carcinoma measurements were significantly higher than from surrounding healthy tissue (p-value <0.0001). Tumor tissue could be detected with a sensitivity of 99% and a specificity of 92% using a cut-off water content value of 69%. Because the Raman measurements are fast and can be carried out on freshly excised tissue without any tissue preparation, this finding signifies an important step towards the development of an intra-operative tool for tumor resection guidance with the aim of enabling oncological radical surgery and improvement of patient outcome.