Abstract A sensitive and reliable assay has been developed to directly screen DNA-targeted anticancer drugs in vitro via using resonance light scattering (RLS) technique. The results of experiments displayed that the increment of RLS intensity was directly proportional to the antitumor effect of anticancer drugs. Through the RLS spectra, the activities of four drugs have been demonstrated as mitoxantrone (MIT) > epirarubicin (EPI) > daunorubicin (DAU) > adriamycin (ADM). However, to further verify the activities of the above four drugs, binding constant ( k) for each of them has been calculated by RLS technique as follows: k RLS (MIT, 8.75 × 10 5 L mol −1) > k RLS (EPI, 6.58 × 10 5 L mol −1) > k RLS (DAU, 4.79 × 10 5 L mol −1) > k RLS (ADM, 3.82 × 10 5 L mol −1). Also, this RLS assay result was validated by seasoned vitro screening methods for anticancer drugs. In all, the proposed RLS is not only a simple, sensitive, objective and straightforward method, but also it is an unprecedented assay for primarily screening DNA-targeted anticancer drugs.