Vaccines containing inactivated toxins confer protection by eliciting a neutralizing antibody response against bacterial toxins such as tetanus and diphtheria. At present, release of tetanus toxoid (TT) and diphtheria toxoid (DT)-containing vaccines relies on in vivo experiments showing the protective vaccine response. The aim of this study was to develop a reliable in vitro assay for TT vaccine antigen characterization with the potential of replacing in vivo potency experiments. To this end, we exploited that TT elicits a recall response in vaccinated donors: human peripheral blood mononuclear cells (PBMC) were stimulated with alum-adsorbed TT bulk antigen and low concentrations of TLR9 ligand; induction of TT-specific IgG was quantified via ELISpot after 5 days. Proof-of-concept was obtained using paired samples from donors before and after vaccination; anti-TT IgG was only detected in PBMC collected after booster vaccination; specificity was demonstrated with DT stimulation as control. Notably, when using PBMC from buffy coats, the specific response to TT was reproducible in 30% of cells; responsiveness correlated with higher numbers of switched memory B cells. Consecutive results showed that TT-specific IgG was also detectable when PBMC were stimulated with DTaP final vaccine product. Thus, the assay provides a viable means to test B-cell differentiation and induction of TT-specific IgG secretion using bulk antigen and final vaccine. However, prequalification of PBMC is required for reliable performance. Along with physicochemical and immunochemical methods, the functional assay could represent a complementary tool to replace in vivo potency assays in batch release of TT-containing vaccines.