Anthracyclines are one of the most studied anticancer drugs approved for medical treatment. The equilibrium constant (K) of the reaction between these drugs with DNA in both in vitro and in vivo experiments lacks consensus. The K values vary from 104 up to 108 M-1, which suggest a 1000-fold error in determining the effective concentration needed to form the drug-DNA complex. Until 2014, only one study by García [J. Phys. Chem. B, 2014, 118, 1288-1295] showed that the binding of anthracycline representative doxorubicin occurs in two reactions. We support this result by brightness analysis at a single molecular level for the four most common anthracyclines: doxorubicin, daunorubicin, epirubicin, and idarubicin.