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For many infectious diseases T cells are an important part of naturally acquired protective immune responses, and inducing these by vaccination has been the aim of much research. Here, we describe a protocol for the analysis of vaccine-induced antigen-specific immune responses. For this purpose, cells of whole spleens obtained from vaccinated BALB/c mice were ex vivo stimulated with the antigen incorporated in the vaccine. Evaluation and characterization of vaccine-induced adaptive T cell responses was performed by assaying spleen cell proliferation through radioactive 3[H]-thymidine incorporation and multiplex cytokine analysis of IL-2, IFN-γ and TNFα in supernatants from spleen cell suspensions. This protocol can be very useful as a starting point for assessing vaccine-induced memory T cell populations in pre-clinical studies.