The dentino-enamel junction (DEJ) connects enamel, that covers the outer surface of a tooth, to a thicker underlying dentin. The DEJ is a critical interface that permits joining these materials that have widely dissimilar mechanical properties. AFM-based nanoindentation and Raman microspectroscopy were used to define the width and composition of human molar DEJ. Indentation elastic modulus and hardness of enamel, dentin, and DEJ were determined along lines of indents made at 2 μm intervals across the DEJ. Indents made at maximum loads at each end of the indent lines were used to make visible markers allowing Raman microspectroscopy at 1 μm intervals across the DEJ, while using the nanoindent markers for orientation and location. Functional DEJ width estimates were made based on results from nanoindentation and Raman microspectroscopy. DEJ width estimates ranged from 4.7 (±1.2) μm to 6.1 (±1.9) μm based on hardness and 4.9 (±1.1) μm to 6.9 (±1.9) μm based on modulus. DEJ width based on Raman peak intensity variations were 8.0 (±3.2) μm to 8.5 (±3.1) μm based on the phosphate peak, and 7.6 (±3.2) μm to 8.0 (±2.6) μm for C-H stretching mode. These estimates are in the range of DEJ width estimates reported using nanoindentation.