Streptococcus pneumoniae is a major human pathogen responsible for the majority of bacterial pneumonia cases as well as invasive pneumococcal diseases with high mortality and morbidity. Use of conjugate vaccines targeting the pneumococcal capsule has dramatically reduced the incidence of invasive diseases, and there are active efforts to further improve the conjugate vaccines. However, in children new pneumococcal vaccines can no longer be tested with placebo-based clinical trials because effective vaccines are currently available. Thus, vaccine studies must depend on surrogate markers of vaccine efficacy. Although traditional antibody levels (e.g., ELISA) are useful as a surrogate marker of protection, they have limitations, and a bioassay measuring the capacity of antibodies to opsonize pneumococci has been developed. This opsonophagocytosis assay (OPA) replicates the in vivo mechanism of antibody protection and should therefore better reflect protection by vaccine-induced antibodies. Technical improvements of OPA have made this bioassay rapid, multiplexed, and practical for analyzing small samples including those from children. Strong correlations between ELISA and OPA have been observed in many studies of young children. However, poor correlations have been found in some important clinical situations (such as determination of protection by cross-reactive antibodies) and populations (such as elderly adults and immunodeficient patients). In these settings, OPA has become a useful supplementary measure of pneumococcal vaccine immunogenicity. Current efforts to standardize OPA will further expand its uses.