By means of ELISA, confocal microscopy, FACS, 2-DE, ImageJ software, MALDI-ToF-MS, and PMF, in this study we report the differentially phosphoproteins expressed in untreated as well as in AZT-treated K562 cells. As evidenced by ELISA, and partly confirmed by confocal microscopy and flow cytometry analysis, an overall increase in Ophosphorylation was found in the AZT-treated samples. Additional analyses allowed to identify 17 spots representing 10 phosphoproteins differentially expressed, proteins which are involved in many significant biological functions such as mRNA processing, assembly and/or transport of ribosome, protein folding, energetic metabolism, cytoskeleton motility, growth control, hypoxia tolerance, allergic and stress responses. Five out of 10 phosphoproteins were up-regulated in K562 cells previously exposed to 20 μM AZT for 3 h (i.e. calreticulin, stathmin, triosephosphate isomerase, stressinduced- phosphoprotein-1, peptidyl-prolyl cis-trans isomerase A). On the contrary, the other five phosphoproteins were down-regulated (i.e. nucleophosmin, lactoylglutathione lyase, 3-hydroxyacyl-CoA dehydrogenase type-2, heterogeneous nuclear ribonucleoproteins A2-B1, alpha-enolase). The proteins identified in the present study represent the first clear report of differential phosphoproteins expression upon AZT treatment of human chronic myeloid leukemia (K562) cells. Therefore, this type of proteomic analysis could be envisaged as a further tool useful for monitoring response(s) to the drug, like the toxic side effects observed in HIV-infected patients under AZT therapy.