Immune response following subsequent encounter of viruses (and vaccines) relies largely on the pool and frequencies of antigen-specific memory B cells. In addition to antibody titers, the reliable measurement of these cells in human blood (peripheral blood mononuclear cells/PBMCs or purified B cells) provides valuable information on the preparedness of the adaptive immune system to respond to infection or vaccines, and potentially supports the discovery of new quantitative correlates of protection. The Mayo Clinic Vaccine Research Group has developed and optimized a high-throughput ELISPOT-based assay for the quantification of influenza A/H1N1 virus-specific memory B cells in human PBMCs. Here, we present the materials and detailed methodology for using this assay on cryopreserved cells for the measurement of recall humoral immunity readiness (antigen-specific memory B cell frequencies) after influenza vaccination. This assay could be readily adapted to other influenza virus strains and other respiratory viruses and vaccines for use in systems biology and larger population-based studies.