Levels of D-aspartic acid (D/L ratio) in cranial non-collagen proteins (acid-soluble peptide fractions) have been reported to increase with age. We isolated total amino acid fractions from the femur and separately isolated acid-insoluble collagen fraction and acid-soluble peptide fractions; then D/L ratios were measured from each fraction by gas chromatography. We evaluated the applicability of their D/L ratios for age estimation based on their correlation coefficient. A sex-related difference was observed in the D/L ratio. In particular, aged females showed a low ratio, suggesting an association with bone disorders. In males, the D/L ratios of acid-soluble peptide fraction showed the highest correlation rate (r = 0.969) with age, and those of total amino acid fraction showed the highest correlation rate (r = 0.633) with age in females. Without separation of male and female, the D/L ratios of total amino acid fraction showed the highest value (r = 0.853). The D/L ratio of acid-soluble peptide fractions differed according to the size of bone powder particles, being higher for larger particle sizes. These results suggest that the application of D/L ratio from total amino acid fraction is the most effective method for estimating age using the human femur. However, care is necessary when studing cadavers that might be females.