Interference between antibodies generated by a combination hepatitis A and B vaccine was investigated by evaluating the quantity and quality of anti-hepatitis A virus (HAV) and anti-hepatitis B surface antigen (HBs) antibodies generated by Twinrix (Hepatitis A Inactivated and Hepatitis B (Recombinant) Vaccine). The magnitude of the immune response was determined by a retrospective analysis of eight clinical trials, completed during stepwise development of Twinrix. The functionality of anti-HAV was evaluated by comparison of routine ELISA results with neutralization assays and was further characterized by defining the epitope-specificity of binding. Functionality of the anti-HBs response was not tested because a validated assay was not developed at the time this study was conducted. Results of all analyses demonstrated that the combination vaccine induced high antibody titers against hepatitis A and B and a functional anti-HAV response, with no evidence of immune interference to either viral antigen.