Monitoring the immune response is an essential aspect of numerous clinical vaccination trials in order to evaluate the efficacy. In these clinical vaccination trials, peripheral blood mononuclear cells (PBMC) are isolated at different time points from patient blood samples and subsequently cryopreserved to allow batch analysis at a later time point. Here, we present a newly developed short-time assay which allows direct ex vivo analysis of multi-epitope antigen-specific immune responses using mRNA electroporation of cryopreserved PBMC. This novel method is a rapid and elegant tool and will be convenient for monitoring the cellular immune status of patients in clinical vaccination settings.