Microtubules are composed by α- and β-tubulin polypeptides. α-tubulin undergoes a reversible posttranslational modification whereby the C-terminal tyrosine residue is removed (Glu-tubulin) and re-added (Tyrtubulin). Recent studies have shown that α-tubulin tyrosine residues can be nitrated and the incorporation of NO2Tyr into the C-terminus of Glu-tubulin forms a complex that blocks the tyrosination/detyrosination cycle, an event that can compromise protein/enzyme functions, such as cell division. Since many studies demonstrated that Glu-tubulin levels increase in cancer, the aim of the present study was to investigate the effect of new drugs, fluorazone derivatives (K1-K2-K9-K10-K11), on the proliferation of melanoma cells. Our results demonstrated that these drugs, except for K2, were able to inhibit cellular proliferation without exhibiting cytotoxicity. The anti-proliferative effect was accompanied by the decrease of Glu-tubulin levels and the increase of its nitration. This effect seems to be a consequence of NO2 induction and NO2Tyr ligation to Glu-tubulin. Collectively, these results, showing that the fluorazone derivatives, by promoting NO2Tyr incorporation into α-tubulin, are able to arrest the cycle of detyrosination/tyrosination and to inhibit cell proliferation, offer new perspectives for the possible usage of these drugs, alone or in combination, as non-toxic, anti-proliferative agents in melanoma.