A custom-made voxel counting software for calculating the ratio between pulp canal versus tooth volume based on cone-beam CT tooth images was developed and evaluated. The aim of this study was to attempt establishing a correlation between the chronological age of a certain individual and the pulp/tooth volume ratio of one of the teeth. Twenty-eight single rooted teeth of 19 individuals with well-known chronological age were scanned by the cone-beam CT (3D Accuitomo, J. Morita, Kyoto, Japan). Next the images were analyzed using the custom-made software. Linear regression analysis was performed. The results of the analysis showed a moderate correlation between the pulp/tooth volume ratio and biological age with a coefficient of determination (R2) of 0.29. Although the present work was limited to a pilot study, the developed technique showed promising results for dental age estimation in a non-invasive manner using cone-beam CT images in living individuals.