Doxorubicin (DOX) is a widely used classical broad-spectrum anticancer drug. The major mechanism of DOX-mediated anticancer activity at clinically relevant concentrations is believed to be via DNA double-strand breaks due to topoisomerase IIα. However, other mechanisms by which DOX causes cytotoxicity have been proposed, including formaldehyde-dependent virtual interstrand cross-linking (ICL) formation. In this study, a method was established whereby cytotoxicity caused by virtual ICL derived from DOX is turned on and off using a cell culture system. Using this strategy, DOX-mediated cytotoxicity in Fanconi anemia group gene (FANC)/breast cancer susceptibility gene (BRCA)-deficient cells increased up to 70-fold compared to that in cells proficient in DNA repair pathways by increasing intracellular formaldehyde (FA) concentration. This approach also demonstrated that cytotoxicity introduced by DOX-mediated FA-dependent virtual ICL is completely independent of the toxicity induced by topoisomerase II inhibition at the cellular level. The potential of dual-targeting by DOX treatment was verified using an acid-specific FA donor. Overall, anticancer therapy targeting tumors deficient in the FANC/BRCA pathway may be possible by minimizing DOX-induced toxicity in normal cells.