Introduction: Breast cancer is the most diagnosed type of cancer in women and the leading cause of death by cancer worldwide. In recent years, active immunotherapy using vaccines has been raised as a novel approach to conventional treatments. Peptide-based vaccines are developed using overexpressed proteins in the tumor, commonly known as tumor antigens, that can stimulate the humoral immune response. Objective: To evaluate in Balb/c mice the production of antibodies induced by inoculation with doses of 30, 50, and 100 µg of the multi-peptide-based vaccine prototype derived from tumoral antigens. Material and methods: The vaccine prototype included the peptides derived from the following proteins: mammaglobin-α, NY-ESO-1, PLAC-1, Syntenin-1, and MAGE-A3. These were selected by in silicio analysis. Peptides were mixed with Freund incomplete adjuvant and inoculated subcutaneously during days 1, 15, 45, and 60 in twelve Balb/C mice classified into four experimental groups (three experimental groups and a placebo group). The mice were bled during days 0, 40, and 80, and serum samples were used to detect the presence of antibodies (IgM, IgG1, IgG2a, IgG2b, and IgG3) and the recognition of each individual peptide by assays of Indirect ELISA and Dot blot. Results: We observed the production of IgM and subclasses of IgG in the three experimental groups (30, 50, and 100 µg), mainly the subclasses IgG2a and IgG2b. In the Dot blot, we observed that immunized mice produce antibodies against all the peptides; particularly, the peptides derived from Syntenin-1, PRAME, mammaglobin-α, and PLAC-1 were the most immunogenic. Conclusion: The multi-peptide-based vaccine prototype induced antibody production against each peptide. The results can contribute to the development of future in vivo experiments focused on the effect of the administration of peptides on avoiding tumoral growth.