Epitopes of regulatory T cells (tregitopes) represent linear sequences of amino acids that induce CD4+CD25+Foxp3+ T lymphocytes expansion both in vitro and in vivo. The tregitopes' effectiveness was confirmed in autoimmune disease mouse models and in murine transplant models. Therefore, tregitopes together with regulatory T cells (Tregs) could play a major role in maintaining immune tolerance. The purpose of the presented study was a selection of potential tregitopes and assessment of their impact on Tregs expansion. Eight peptides were selected based on the previously published in silico model and their immunotolerogenic functions. To verify, if selected peptides are potential TCR ligands, the affinity of selected peptides to overrepresented in patients with autoimmune diseases, HLA-DRB1*04:01 allele, was measured by surface plasmon resonance. In order to evaluate the impact of potential tregitopes on the induction of Tregs in in vitro conditions, C57BL6Foxp3GFP mouse antigen presenting cells were co-cultured with naive syngeneic T cells under stimulation of selected peptides. CD4+CD25+Foxp3+ and CD4+CD25+Foxp3+IL-10+ cells frequency was analyzed using flow cytometry. Based on Tregs induction, two tregitopes derived from yeast and adenovirus protein were identified. In summary, the performed studies allowed an identification of novel putative tregitopes, which application potential includes their use as immunomodulators in mice.