In forensics, the positive identification of decomposed or skeletonized bodies is a fundamental task, with the age-at-death estimation of adult individuals as one of the main objectives. Among different dental methods, root dentin translucency (RDT) is often applied since it is easy to perform and non-destructive. However, this method has some biases, and several equations have been proposed in the literature. This study aimed to test the performance of the previously published equations in an Italian sample with known age and sex, and to develop an equation specific to the Italian population. In total, we examined a sample of 155 single and multi-rooted teeth from male and female individuals aged 18-85 years. The regression equation developed for Italians was tested on a holdout sample drawn from the same population. Intra- and inter-observer errors were calculated using ICC analysis. Both root length and RDT showed excellent repeatability and reproducibility regardless of tooth type. Two of the seven published equations tested performed better in our sample, but the newly proposed equation performed better than those on the Italian population. In conclusion, RDT has proven to be a reliable indicator for age estimation, and the proposed new formula may be effective in such estimation, especially in individuals aged <40.