The aim of this study was to perform a structural characterization of dental pulp tissue using multiphoton microscopy and to compare this to classical histological coloration. Materiel & Method: Sample preparation: Freshly extracted teeth with healthy pulp were used in this study. All teeth were fixed in PFA 4% for 24 hours, rinsed in PBS and decalcified. The samples were then embedded in paraffin. The paraffin blocks were cut with a Microm HM340E microtome with Niagara system to obtain 3 μm thick sections. The samples were then dewaxed with xylene, rehydrated and stained with Hematoxilin/eosin and Masson’s trichrome. Multiphoton Microscopy (MPM) images were recorded using a custom-built multiphoton microscope based on a SliceScope microscope (MPSS-1000P, Scientifica) upright microscope. MPM images were obtained recording the non- linear emission spectra after laser scanning of samples. Two-photon fluorescence (2-PEF) signal was used to image autofluorescent structures. Second harmonic generation signal (SHG) was performed to image collagen. The recorded images were processed by ImageJ software. Results: At the dentin/pulp junction, we observed the dentin, which emitted an important SHG signal due to the abundance of type 1 collagen fibers. The palisade of odontoblasts and odontoblast extensions (2PEF) were imaged towards the dentinal tubules. We imaged blood vessels due to the 2-PEF signal of blood cells. A fibrous structure representing the extracellular matrix emitted a SHG signal, showing the presence of type I collagen fiber. At the apical 1/3 of the root fibers were observed at the periodontal ligament emitting both in SHG and 2PEF, coming in contact to the pulp. The MPM images were then compared to the histological stained samples. Conclusion: Multiphoton microscopy is a convenient label-free minimally invasive technique to image live, vascularized and dynamic tissue such as the pulp. It provides valuable additional information with respect to histological sections observed under an optical microscope.