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Raman Difference spectroscopy (RDS) presents a promising, non-invasive approach for facilitating rapid diagnosis of oral squamous cell carcinoma (OSCC). Many ex vivo studies rely on formalin-embedded tissue specimens. Spectroscopic changes and classification of formalin-fixed OSCC tissues were investigated. In an analysis of 47 specimens (n = 30 OSCC, n = 17 physiological mucosa), RDS was employed at 180 distinct measurement loci after storage in saline, and again at 125 measurement loci after storage in formalin. For classification, Principal Component Analysis (PCA) and Linear Discriminant Analysis (LDA) were utilized. In distinguishing OSCC from physiological mucosa, LDA revealed a higher sensitivity (97.6% for NaCl-stored vs. 90.9% for formalin-fixed tissues), specificity (93.0% vs. 78.6%), and overall classification accuracy (95.2% vs. 85.4%) for specimens stored in saline. While formalin fixation is valuable for tissue preservation in histopathology, Raman spectra of OSCC tissues were substantially altered, with implications for validation of innovative optical techniques such as Raman Spectroscopy.