Complete characterization of the B cell response to infection or vaccination is dependent on accurate quantitation of the memory B cell (MBC) pool. An established method for measuring MBC frequencies is limiting dilution analysis based on in vitro stimulation of MBCs to divide and differentiate into antibody-secreting cells (ASCs). The presence of specific antibody then serves to identify cultures positive for precursor MBCs. The sensitivity of this approach is critically dependent on optimal in vitro MBC activation. To develop a limiting dilution assay (LDA) for measuring influenza-specific MBC frequencies, we evaluated strategies for the in vitro stimulation of influenza-specific MBCs. An ELISPOT assay to enumerate influenza-specific IgG ASCs was used as the readout for MBC activation. Culture of influenza-specific MBCs with influenza-infected splenocytes was effective for MBC activation, but T cell-associated factors were required for optimal LDA sensitivity and clonal expansion of activated MBCs. However, optimal influenza-specific MBC activation was T cell-independent when MBCs were simply cultured with beta-propiolactone (BPL)-inactivated influenza virus particles (BPL-flu). BPL-flu did not stimulate naïve B cells to produce influenza-specific IgG, demonstrating that only MBCs were activated. In addition, BPL-flu acted selectively and only activated influenza-specific MBCs, not MBCs of other specificities. Analysis of influenza-specific MBC frequencies in different anatomical locations in influenza-immune mice established that in vitro stimulation with BPL-flu provided the basis for a sensitive and reproducible LDA. Extending our studies to the herpes simplex virus (HSV) system, we demonstrated that HSV-specific MBCs cultured with BPL-inactivated HSV were selectively activated to IgG secretion in the absence of T cells. Our studies identify BPL-inactivated viral particles as a valuable tool for selective, T cell-independent activation of virus-specific MBCs in vitro. This strategy eliminates the influence of poorly defined T cell-associated factors on MBC frequency determinations.