The challenge of malaria vaccine development is to find multi-stage antigens conserved between Plasmodium species. Our group recently demonstrated that P. vivax (Pv)-infected reticulocytes can activate cytotoxic T lymphocytes (CTLs), in an antigen-dependent manner, describing a protective mechanism against blood-stage parasites. In unpublished data, we performed an immunopeptidomic analysis to identify HLA-I-associated peptides of Pv-infected reticulocytes (iRetics). Most eluted peptides are derived from 'housekeeping' proteins, which are conserved in different Plasmodium stages and species. We selected 50 peptides and their source proteins to be studied as vaccine candidates. P. yoelii (Py) infection is an excellent experimental model for investigating malaria immune response. First, we tested the 50 peptides in an IFNg ELISPOT assay using Py-infected mice splenocytes. To the most relevant peptides, we generated antigen-specific CTLs to perform a killing assay against iRetics. Hence, we produce three Pv recombinant proteins (L3, S2, and H2) in the E. coli expression system for the immunization protocol. Mice were immunized three times with recombinant protein associated with Alum + CpG as an immunological adjuvant. Twenty-one days after the last boost, mice were challenged with 106 PyX17NL-infected red blood cells, and the parasitemia was monitored for 30 days. We identify 23 of the 50 Pv peptides were immunogenic in the Py acute infection. Moreover, 12 remain in the convalescent phase, suggesting memory response. We described that Pv antigen-specific CD8 cells were able to specifically kill Py-iRetics, which was not observed with unstimulated CD8 cells or uninfected reticulocytes. Regarding the immunization assessment, we identify that 2 of 3 tested proteins can induce antigen-specific IgG Total, IgG1, and IgG2c levels, additionally to decrease up to 80% the parasitemia compared to the control group. Therefore, we unprecedentedly identify Pv antigens that can induce a protective response to Py infection, indicating cross-species protection and potential vaccine candidates.