The agents commonly used in the cancer treatment present diverse side effects, and another problem observed during the treatment is the resistance acquisition of the cancer cells against non-related structurally compounds. The aim of this work was to use riboflavin to improve the antineoplastic activity of traditional drugs in the leukemia treatment. HL60 cells were treated with a combination of mitoxantrone (25nM-1000nM) plus riboflavin (20mM) for 4h, 12h and 24h. The effect of the combination was evaluated by MTT reduction and western blot. We observed that the combination of mitoxantrone plus riboflavin caused an expressive decrease of the antineoplastic agent dose and of the period of cells exposition required for producing cytotoxic effect. We also observed cell cycle arrest and apoptosis, as demonstrated by downregulation of c-myc and upregulation of TNFR1. In addition, the combination effect was abolished by a protein phosphatase inhibitor (PP2A). Our results suggest that association of mitoxantrone plus riboflavin could increase the drug availability in the cellular target.