p53-Based cyclotherapy is proving to be a promising approach to palliate undesired effects of chemotherapy in patients with tumours carrying p53 mutations. For example, pre-treatment of cell cultures with Nutlin-3, a highly-selective inhibitor of the p53-mdm2 interaction, has been successfully used as a cytostatic agent to protect normal cells, but not p53-defective cells, from subsequent treatment with mitotic poisons or S-phase specific drugs. Here we sought to evaluate whether low doses of Actinomycin D (LDActD), a clinically-approved drug and potent p53 activator, could substitute Nutlin-3 in p53-based cyclotherapy. We found that pre-treatment with LDActD before adding the aurora kinase inhibitor VX-680 protects normal fibroblasts from polyploidy and nuclear morphology abnormalities induced by VX-680. However, and although to a lower extent than normal fibroblasts, tumour cell lines bearing p53 mutations were also protected by LDActD (but not Nutlin-3) from VX-680-induced polyploidy. We also report that a difference between the response of p53 wild-type cells and p53-defective cells to the LDActD/VX-680 sequential combination is that only the former fail to enter S-phase and therefore accumulate in G1/G0. We propose that drugs that incorporate into DNA during S-phase may perform better as second drugs than mitotic poisons in cyclotherapy approaches using LDActD as a cytostatic agent.