Doxorubicin (DOX) ranks among the most effective anticancer agents. Increasing the formation of covalent DOX-DNA interstrand cross-links can improve the anticancer activity of DOX. However, due to the low stability of the DOX-DNA cross-links to heat and alkali, DOX can be extensively lost during isolation procedures of biochemical methods, thus reducing the apparent clinical relevance of this mechanism. Here, we developed a drug label-free, single-molecule magnetic tweezers assay that can detect a single DOX-DNA cross-link on the basis of the significant increase of the unzipping forces of DNA hairpins upon drug binding. Using this assay, we measured the DOX concentration-dependent cross-linking rates at clinically relevant concentrations of DOX. We report an ∼26-fold higher formaldehyde concentration dependence of cross-linking rates than previously reported and 0.9 ± 0.8 cross-links/10³ bp at the clinically relevant concentrations of 70 nM DOX and 50 μM formaldehyde. Our results suggest a much higher cross-link formation ability than previous bulk measurements have reported and suggest that the cross-linking mechanism has promising therapeutic potential. This general method can be used to detect the formation kinetics of other DNA lesions or DNA adducts that affect DNA duplex stability.