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In this preliminary study, a multidisciplinary method based on high-resolution analytical techniques (such as microcomputed tomography, Raman Microspectroscopy, scanning electron microscopy, and Vickers microhardness test) was exploited to evaluate the alterations that occur in human teeth at the initial stage of the carious lesion. To this purpose, six extracted molars displaying a natural white spot lesion (WSL) were investigated. Specific morphological, structural, and chemical parameters, such as the mineral density, indentation hardness, molecular and elemental composition, and surface micromorphology were obtained on the WSL, and the results were statistically compared (t-test, p < 0.05) to those of the sound enamel on the same tooth. In the WSL, with respect to the sound area, a decrease in the mineral density and crystallinity was detected together with differences in the molecular composition and surface microstructure, such as the occurrence of micropores and irregularities. Moreover, the elemental analysis highlighted in WSL showed a statistically significant decrease in Ca and P percentages. In conclusion, this multidisciplinary approach allows us to fully characterize the area of interest, providing a deeper knowledge of these enamel lesions, which could have important clinical implications. [ABSTRACT FROM AUTHOR]