Dentin carious lesion is a dynamic process that involves de- mineralization and collagen denaturation. Collagen type I is the major protein in dentin and it has been investigated based on its optical properties. Multiphoton microscopy (MPM) is a nonlinear imaging technique that reveals the car- ies process using the collagen two-photon excitation fluo- rescence (2PEF) and its second-harmonic generation (SHG). Combining the histological and the International Caries De- tection and Assessment System (ICDAS) classifications with nonlinear optical spectroscopy (NLOS), 2PEF and SHG inten- sities of enamel and dentin were highly altered during the caries process. It has been proven that the ratio SHG/2PEF is a relevant indicator of the organic matrix denaturation [Ter- rer et al.: J Dent Res 2016;96:574–579]. In the present study, a series of measurable signals is made to detect early stages of carious lesion according to the ICDAS classification and to explore the relationship between these measures and the ICDAS scale. Comparison of the efficiency of nonlinear opti- cal signals for caries detection with the ICDAS classification is essential to evaluate their potential for clinical applica- tion. In our study, the use of the NLOS measured by MPM allowed us to monitor a quantitative parameter (SHG/2PEF ratio) according to the dentin carious lesion state (ICDAS and histological examination). Three coherent new groups were defined (ICDAS 0/1; ICDAS 2/3; ICDAS 4/5/6), where the carious process can be clearly described with a statistically significant decrease of the SHG/2PEF ratio.