The deoxyribonucleic acid (DNA) is the molecule of life that controls all the chemical changes that take place in cells. It is the molecular target for many of the drugs that are used in cancer therapeutics, and is viewed as a non-specific target of cytotoxic agents. The interaction of drugs with DNA is among the most important aspects of biological studies in drug discovery and pharmaceutical development processes. Moreover, the knowledge of specific targets in rational design of chemotherapeutics is a fundamental factor, principally, for the design of molecules that can be used in the treatment of oncologic diseases. Observing the pre- and postsigns of drug-DNA interaction provides good evidence for the interaction mechanism to be elucidated. Also, this interaction could be used for the quantification of drugs and for the determination of new drugs targeting DNA. Approaches can provide new insight into rational drug design and would lead to further understanding of the interaction mechanism between anti-cancer drugs and DNA. The intention of this review is to study of anticancer drugs, DNA interaction, and the mechanisms of interaction in order to understand the influence of several interaction factors in the capacity and selectivity of the anticancer drugs to interact with DNA. In addition, different experimental approaches to detect and to evaluate the anticancer drugs’ interactions with DNA were also mentioned.