A challenging and crucial component to clinical caries diagnosis is diagnosing the activity of early lesions because it may have a significant impact on treatment decisions. This study aims to utilize microtomographic (micro-CT) scans of the enamel smooth surface layer to serve as a reference standard for future ex vivo caries activity assessment validation studies. Sound and non-cavitated (ICDAS 1-3) smooth surfaces (n=59) of extracted permanent teeth were examined for caries activity by calibrated individuals via visual-tactile examination. Each surface was scanned via micro-CT and line plot analysis generated plots of the mineral density against lesion depth, where the AUC was calculated. AUC thresholds were established to classify sound, remineralized, and demineralized surfaces against the gold standard examiner’s ICCMS assessment of sound, inactive, and active lesions, respectively. Diagnostic thresholds were associated with caries ICCMS activity classification using AUC mineral density values of the most external 96 μm of enamel. The established thresholds demonstrated 76.3% agreement with the ICCMS assessment in identifying demineralized lesions (k=0.45), with high sensitivity (0.73) and specificity (0.77). This study demonstrates quantifiable differences among demineralized, remineralized lesions, and sound surfaces, which contributes to the establishment of micro-CT as a reference standard for caries activity that may be used to improve clinical and technological caries examinations.