Tumor suppressor protein 53 plays an important role in the initiation of cell cycle arrest and apoptosis. Being highly mutated in several different cancer types, p53 is a good target for anticancer therapeutics. It has been shown that a peptide derived from the C-terminus of p53 activates specific DNA-binding of endogenous mutated p53, restoring its original activity. Detection of short cell-penetrating peptide sequences using quantitative structure-activity relationship algorithm gives new opportunities for developing novel peptide-based platforms for modulation of biological activity inside the cell. Here we present novel human protein 53 C-terminal domain-derived peptides, Peptide4 and Peptide5 that were designed using cell-penetrating peptide prediction algorithm and synthesised by Fmoc chemistry. Peptide4 and Peptide5 showed to be capable for translocation inside the breast cancer cells. Subsequent introduction of stearic acid moiety in the backbone of these peptides at N-terminal or lysine 3-orthogonal positions enhanced their cell-penetrating ability. Moreover Peptide4 and Peptide5 showed certain cytotoxic activity and were able to induce apoptosis in MDA-MB-231 cell line in the absence of serum. We suggest that human protein 53 C-terminal domain-derived cell-penetrating peptides Peptide4 and Peptide5 have promising perspectives for the future anticancer applications.