Objective: This study aimed to evaluate the antibody responses in two adult age groups after diphtheria vaccination. Study Design: An observational analytic study was carried out to determine the difference in serum titer of anti-diphtheria antibody. Methods: Serum antibody titers were measured just before and 3 months after injection of Diphtheria toxoid vaccine. Vaccine was given to two adult age groups of health care personnel in hospital: the young (< 40 years) and the middle-aged (≥ 40 years). Data were analyzed using the Mann-Whitney test (p < 0.05). Results: Significant increase in serum anti-diphtheria antibody titers were recorded after vaccination in both age group (p < 0.001 in young adult and p = 0.001 in middle-aged adult, respectively). There were no substantial differences between the two groups in terms of antibody titer before vaccination (p = 0.741), 3 months after vaccination (p = 0.317) and in the increase of antibody titer (p = 0.479). Conclusions: This study showed that there was no significant difference in the increase of anti-diphtheria antibody titers between the two age groups, proving that both young and middle-aged adults had an equal immune response to a given diphtheria vaccine.