During recent years, vaccination against hepatitis A has been implemented in several countries. It is expected that the increase in mass vaccination against hepatitis A will eventually result in a decreased prevalence of anti-HAV antibodies in the general population. For this reason, a suitable clinical sample for diagnosis of hepatitis A must be sufficiently sensitive to enable detection of lower antibodies titers. In this study, the feasibility of using dried blood spots (DBS) was assessed for the detection of anti-HAV antibodies after a natural infection and vaccination. Seventy-four DBS and paired plasma samples were obtained from a group of college students for a cross-sectional hepatitis A seroepidemiological study. Forty-six students seronegative for anti-HAV were selected randomly and immunized with an inactivated hepatitis A vaccine using an 0-6 month schedule. Seroconversion was monitored in paired plasma and DBS samples 6 months after the first dose followed by a period of 8 and 24 months after the second dose. A strong correlation between OD/CO rates of paired plasma and DBS samples for the detection of anti-HAV was observed. The sensitivity and specificity of the DBS compared with plasma for the detection of anti-HAV antibodies after natural infection was 100%. The sensitivity of DBS in samples collected 24 months after the second dose of hepatitis A vaccine was 95.4%. The results showed that DBS samples can be used for the detection of anti-HAV antibodies both after natural infection or vaccination.