The enzyme-linked immunospot (ELISPOT) assay is a widely used tool for enumeration of antigen-specific memory B cells in several disciplines, such as vaccination, cancer immunotherapy and transplantation. For the accurate estimation of antigen-specific memory B cell frequencies, a well-defined B cell activation protocol is pivotal. In this study, we aimed to characterize a polyclonal B cell activation protocol to facilitate optimal monitoring of antigen-specific memory B cell frequencies. Total, naive and memory B cells were activated polyclonally with an α-CD40 monoclonal antibody, cytosine–phosphate–guanine (CPG) oligodeoxynucleotide (ODN) 2006, interleukin (IL)-2, IL-10 and IL-21. Polyclonal activation of B cells resulted in equal cell death ratios in naive and memory B cells. When tested in an antigen-specific system, immunoglobulin (Ig)G spots were detected only in the memory fraction. There was no change in B cell polyclonality due to in-vitro activation. Our data show that the current polyclonal activation protocol may be used reliably to estimate the frequency of memory B cells in ELISPOT assays.