Studying the interaction of antitumor-active anthraquinones with biologically important redox couples is important in understanding the possible reductive or oxidative mode of metabolism of these antineoplastic agents coupled with the formation of free radicals. The interactions of such anthraquinones, i.e., carminic acid (CA) and mitoxantrone (Mx) with iron(II, III) and copper(I, II) redox couples in oxygenated and deaerated solutions, were investigated by UV-Visible and IR-spectroscopy. The superoxide radical reagent, nitroblue tetrazolium (NBT), was added to the metal and anthraquinone solutions and their binary mixtures at varying pH. Formazan, the reduction product of NBT, was produced mainly as a result of Fe(II)-NBT and Fe(II)-Mx-NBT interactions. The ternary mixtures of the lower valencies of iron and copper with CA and NBT exhibited intensive charge-transfer bands in the visible region, while metal-Mx-NBT combinations did not produce such bands, possibly due to the blockage of the redox-active aminoethanolamine side-chains of Mx through coordination with the metals. Copper-Mx combinations showed an oxygen sensitivity as spectral evidence was obtained for the oxidative transformation of Mx to the cyclic primary metabolite. The results were evaluated in regard to the possible oxidative activation of the studied anthracenediones with iron and copper systems.