AbstractThe main focus of this study is exploring the effect and mechanism of two HIV‐protease inhibitors: Ritonavir and Ritonavir‐nitric oxide (Ritonavir‐NO) on in vitro growth of melanoma cell lines. NO modification significantly improved the antitumor potential of Ritonavir, as the IC50 values of Ritonavir‐NO were approximately two times lower than IC50 values of the parental compound. Our results showed for the first time, that both compounds induced senescence in primary and metastatic melanoma cell lines. This transformation was manifested as a change in cell morphology, enlargement of nuclei, increased cellular granulation, upregulation of β‐galactosidase activity, lipofuscin granules appearance, higher production of reactive oxygen species and persistent inhibition of proliferation. The expression of p53, as one of the key regulators of senescence, was upregulated after 48 hours of Ritonavir‐NO treatment only in metastatic B16F10 cells, ranking it as a late‐response event. The development of senescent phenotype was consistent with the alteration of the cytoskeleton—as we observed diminished expression of vinculin, α‐actin, and β‐tubulin. Permanent inhibition of S6 protein by Ritonavir‐NO, but not Ritonavir, could be responsible for a stronger antiproliferative potential of the NO‐modified compound. Taken together, induction of senescent phenotype may provide an excellent platform for developing therapeutic approaches based on selective killing of senescent cells.