Among the many nucleoside analogues, 3’-azido-2’,3-dideoxythimidine (AZT, zidovudine, Retrovir^®), 2’,3’-dideoxycytidine (ddC, zalcitabine, Hivid^®), 2’,3’-dideoxyinosine (ddI, didanosine, Videx^®), 2’-deoxy-3’-thiacytidine (3TC, lamivudine, Epivir^®), and 2’,3’-dideoxy-5-fluoro-3’-thiacytidine (FTC, emtricitabine, Emtriva^®) are the only ones that as such or in combination with other drugs have been approved by the FDA to treat HIV/AIDS patients. Following our past investigations where we used AZT and K562 cells, in this study different nucleoside analogues such as 5-azacytidine (AzaC), ddC, and ddI were tested exploring, as in the past, their effects on cell viability, cell cycle, and protein phosphorylation/O-GlcNAcylation in the same cell type, i.e. K562 cells. Our results indicate that with respect to ddI, both AzaC and ddC are more effective in inducing a significant distribution and/or a substantial content alteration at the level of phospho-amino acids residues (i.e. O-Ser, O-Thr, and O-Tyr) as well as at the level of O-GlcNAc residues. Moreover, with respect to the other two drugs (i.e. ddC, and ddI) AzaC seems to act in slower times. Eventually, with respect to other similar studies, this paper is the only one which gives some insight upon cell viability, cell cycle, and protein phosphorylation/O-GlcNAcylation when K562 cells are separately exposed to AzaC, ddC, or ddI.