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This study aims to detect high grade squamous intraepithelial cells (HSIL) by investigating HSIL associated biochemical changes in morphologically normal appearing intermediate and superficial cells using Raman spectroscopy. Raman spectra (n = 755) were measured from intermediate and superficial cells from negative cytology ThinPrep specimens (n = 18) and from morphologically normal appearing intermediate and superficial cells from HSIL cytology ThinPrep specimens (n = 17). The Raman data was subjected to multivariate algorithms including the standard principal component analysis (PCA)-linear discriminant analysis (LDA) and partial least squares discriminant analysis (PLS-DA) together with random subsets cross-validation for discriminating negative cytology from HSIL. The PCA-LDA method yielded sensitivities of 74.9%, 72.8%, and 75.6% and specificities of 89.9%, 81.9%, and 84.5%, for HSIL diagnosis based on the dataset obtained from intermediate, superficial and mixed intermediate/superficial cells, respectively. The PLS-DA method provided improved sensitivities of 95.5%, 95.2% and 96.1% and specificities of 92.7%, 94.7% and 93.5% compared to the PCA-LDA method. The results demonstrate that the biochemical signatures of morphologically normal appearing cells can be used to discriminate between negative and HSIL cytology. In addition, it was found that mixed intermediate and superficial cells could be used for HSIL diagnosis as the biochemical differences between negative and HSIL cytology were greater than the biochemical differences between intermediate and superficial cell types.