The growth of plants, animals, and humans can give rise to layered structures associated with annual periodicity. Thickness variations are often correlated to nutrition supply and stress factors. The annual layers in a tree trunk with millimeter thickness can be directly counted, whereas the layers in tooth cementum with micrometer thickness are made visible using optical microscopy. These optical techniques rely on the surface evaluation or thin, optically transparent slices. Hard Xray tomography with micrometer resolution, however, provides a three-dimensional view without physical slicing. We have developed a procedure to enhance the tomography data of annual layers in human and bovid tooth cementum. The analysis of a substantial part of an archeological human tooth demonstrated that the detected number of layers depended on the selected region and could vary between 13 and 27. The related average thickness of the annual layers was found to be (5.4 ± 1.9) µm for the human tooth, whereas the buffalo tooth exhibited a layer periodicity of 46 µm. The present study elucidates the potential of combining computational tools with high-quality micro computed tomography data to quantify the annual layers in tooth cementum for a variety of purposes including age-at-death determination.