A new biochemical method for estimation of dental age from human dentin is presented. An adaptation and modification of a dentin protein extraction protocol was developed by which deoxypyridinoline crosslinks were measured using an enzyme immunoassay technique. Deoxypyridinoline, a non-reducible collagen crosslink, was measured in human dentin samples from permanent individual molars. The measurement of this protein in dentin samples from 22 patients with ages ranging from 15 to 73 years old was carried out to obtain an estimate of age at death. By linear regression between deoxypyridinoline values and age, equations for age calculations were computed. The error between the estimated age and the known age at extraction was calculated. Our results show that this method produces an expected associated error on calibration averaging 14.9 years about the mean estimated values, at a 65% level of confidence. Values of sensitivities and specificities were calculated for deoxypyridinoline at different ages. We conclude that determination of deoxypyridinoline in human dentin extracts is a new and reliable procedure, potentially suitable for most forensic laboratories and which could be a useful tool to estimate human dental age. This method could be considered as an alternative method to calculate dental age, contributing to a more accurate estimation of chronological age when used in addition to other morphological or biochemical dental methods.