ObjectivesFourier Transform Infrared microspectroscopy let characterize the macromolecular composition and distribution of tissues and cells, by studying the interaction between infrared radiation and matter. Therefore, we hypothesize to exploit this analytical tool in the analysis of inflamed pulps, to detect the different biochemical features related to various degrees of inflammation.Materials and MethodsIR maps of 13 irreversible and 12 hyperplastic pulpitis, together with 10 normal pulps, were acquired, compared with histological findings and submitted to multivariate (HCA, PCA, SIMCA) and statistical (one‐way ANOVA) analysis. The fit of convoluted bands let calculate meaningful band area ratios (means ± s.d., P < 0.05).ResultsThe infrared imaging analysis pin‐pointed higher amounts of water and lower quantities of type I collagen in all inflamed pulps. Specific vibrational markers were defined for irreversible pulpitis (Lipids/Total Biomass, PhII/Total Biomass, CH2/CH3, and Ty/AII) and hyperplastic ones (OH/Total Biomass, Collagen/Total Biomass, and CH3 Collagen/Total Biomass).ConclusionThe study confirmed that FTIR microspectroscopy let discriminate tissues’ biological features. The infrared imaging analysis evidenced, in inflamed pulps, alterations in tissues’ structure and composition. Changes in lipid metabolism, increasing amounts of tyrosine, and the occurrence of phosphorylative processes were highlighted in irreversible pulpitis, while high amounts of water and low quantities of type I collagen were detected in hyperplastic samples.