Purpose: Physiological dentin sclerosis is a distinctive phenomenon observed in transverse sections of the tooth root as a tooth ages. Since this effect has only been visualized 2-dimensionally, this pilot proof-of-concept study aimed to propose a novel method to visualize and quantify physiological sclerotic dentin 3-dimensionally using density-based micro-computed tomography (CT)-generated gray values. Materials and Methods: Forty single-rooted premolars of known chronological age from the Malay male population were scanned with a micro-CT scanner. The X-ray source was operated at 160 kV, 100 μA, and a voxel size of 48.8 μm.Three-dimensional volume datasets for each tooth were reconstructed, and volumetric analysis was performed on the root dentin region using the open-source software FIJI (ImageJ) (ver. 1.54k, National Institutes of Health, Bethesda, USA). Results: Micro-CT imaging enabled the selection, isolation, and quantification of high-density areas in the tooth rootthat correspond to physiological sclerotic dentin. The generated gray value profile also reflected the characteristicbutterfly-shaped pattern seen in transverse root sections. A comparison of premolars from different age groups revealedthat individuals aged 31-60 years exhibited a larger butterfly-shaped area, reflecting the increase in high-density sclerotic dentin, compared with younger premolars. Conclusion In this pilot proof-of-concept study, visualization and measurement of physiological sclerotic dentin were shown to be feasible using micro-CT. However, the method is highly dependent on the capability of the micro-CT scanner and the performance of the image-processing software. Further research is needed to improve image quality using advanced micro-CT scanning techniques.