The aim of this cross-sectional study was to evaluate serum levels of the hormone, dehydroepiandrosterone sulphate (DHEAS), during the pre-pubertal, pubertal, and adult stages of skeletal maturation based on the methods of Björk and Grave and Brown of assessing hand-wrist radiographs. The levels of the DHEAS of each individual were measured using quantitative enzyme-linked immunosorbent assay and correlated with the corresponding stages in their hand-wrist radiograph. This study was performed on 60 subjects (30 females and 30 males) aged from 7 to 30 years. Analysis of variance followed by a Tukey honestly significant difference test showed that the serum levels of the DHEAS were statistically significant at (P < 0.01) in all three groups. The serum levels were significant (P < 0.05) when each of the three groups were individually compared with the other two groups. The mean DHEAS levels were 0.43 ± 0.28, 2.17 ± 0.92, and 4.60 ± 1.34 μg/ml in the pre-pubertal, pubertal, and adult groups, respectively. There was a gradual increase in the hormonal level with progressing skeletal age. The adult group showed the highest DHEAS level and the pre-pubertal group the lowest. Serum levels of DHEAS showed a constant increase from pre-puberty to adulthood, and at the same level of skeletal maturation, both females and males had similar hormone levels. This finding highlights the fact that the hormone DHEAS plays a significant role and can be a valuable tool in assessing skeletal maturation.