In our work, the effect of a dental Er:YAG pulsed laser and a diamond cylindrical drill with a turbine handpiece on dentin tissue was studied using spectral imaging. The combination of spectral imaging of FTIR microspectroscopy data and subsequent multivariate analysis (hierarchical cluster analysis (HCA) and principal component analysis (PCA)) was shown to unambiguously detect visually indistinguishable structural changes occurring in the hard dental tissue (dentin) depending on the method used for their pre-processing, and to classify and differentiate the identified features at the submicron level with high spatial resolution. The detectable spectral transformations indicate that the preparation of dental tissue with a dental laser leads to significant changes in the organic components of dentin, which may affect adhesion. The use of a diamond cylindrical drill with a turbine handpiece is characterized by a larger area (depth) of the altered hard tissue than in the case of a dental laser for dental cavity preparation. The observed redistribution of the phase composition of the inorganic component in the tissue is associated with the emergence of additional phases of weak calcium phosphates, and changes in the organic component with transformations in the secondary structure of proteins. Active use of the proposed integrated approach in the future will clarify the areas of its applicability to the analysis of biological tissues and pathologies in them, which will help in the clinical setting to choose the optimal personalized approach for patients.