Photothermal radiometry and modulated luminescence (PTR-LUM) is an emerging nondestructive methodology applied toward the characterization and quantification of dental caries. We evaluate the efficacy of PTR-LUM in vitro to detect, monitor, and quantify human enamel caries. Artificial caries are created in extracted human molars (n = 15) using an acidified gel system (pH 4.5) for 10 or 40 days. PTR-LUM frequency scans (1 Hz–1 kHz) are performed before and during demineralization. Transverse microradiography (TMR) analysis, the current gold standard, follows at treatment conclusion to determine the mineral loss and depth of the artificially demineralized lesions. A theoretical model is applied to PTR experimental data to evaluate the changes in optothermophysical properties of demineralized enamel as a function of time. Higher optical scattering coefficients and poorer thermophysical properties are characteristic of the growing demineralized lesions, as verified by TMR, where the generated microporosities of the subsurface lesion confine the thermal-wave centroid. Enhanced optical scattering coefficients of demineralized lesions result in poorer luminescence yield due to scattering of both incident and converted luminescent photons. PTR-LUM sensitivity to changes in tooth mineralization coupled with opto-thermophysical property extraction illustrates the technique's potential for nondestructive quantification of enamel caries.