Ruthenium complexes are small synthetic molecules with a wide range of uses including cancer therapy, and as a research tool to understand chemical carcinogenesis. Specifically, ruthenium dimers like [?-C4(cpdppz)2(phen)4Ru2]4+ have been engineered to have a high affinity for DNA and a very low dissociation rate. The complex consists of two Ru(phen)2 moieties connected by a flexible linker, and its strong DNA binding inhibits replication of DNA in target cancer cells. To quantify the rate at which DNA binding occurs for this dimer, double-stranded DNA is stretched with optical tweezers, and exposed to the ligand under a fixed applied force. When binding to DNA, the two Ru(phen)2 moieties intercalate between base pairs via a threading mechanism. Intercalation results in an increase in the length of the target DNA, and the rate of this increase depends exponentially on the applied force. Force-dependent fast and slow rates indicate two step binding in which the rate-limiting step of each intercalation event involves DNA elongation by 0.22 ± 0.01 nm. The intercalation of the first moiety is rapid, followed by threading of the complex which is an order of magnitude slower as it requires local DNA melting. These studies demonstrate the capability of optical tweezers to elucidate the mechanism of complex DNA-ligand interactions, which may facilitate the rational design of DNA binding ligands with specific DNA interaction properties.