The majority of antibody binding sites (B-cell epitopes) on antigens are discontinuous. The binding between antigen and antibody is specific, but in some cases, the antibody elicited by one antigen will show cross-reactivity against other antigens. We have developed a bioinformatics-based approach for the analysis of sequence variability of neutralizing antibody binding sites and the assessment of coverage by individual neutralizing antibodies. The antigenic analysis of functional sites on the envelope (E) protein from dengue virus has been used as a case study. The description of B-cell epitopes, measurement of epitope similarity among different strains, and estimation of antibody neutralizing coverage provide insights in antibody cross-reactivity. We have defined a generalized method for the analysis of cross-reactivity of neutralizing antibodies that is also applicable to the analysis of other pathogens. This method adds to the toolset available for the characterization and the design of broadly neutralizing vaccines.