Pemetrexed is a well-known and widely used antineoplastic drug under the category of cytotoxic, folate anti-metabolites that is used in chemotherapeutic treatments, especially in malignant mesothelioma and non-small cell lung carcinoma. Here, the binding mechanism and interactions of Pemetrexed with double strain fish sperm deoxyribonucleic acid (dsDNA) were studied thoroughly both experimentally and theoretically, using multi-spectroscopic techniques and molecular docking simulations. Our ultimate goal is to understand better the potential of such antineoplastic drugs and, hence, to design drugs with high dsDNA binding affinities and fewer adverse effects. We employed several techniques yielding different but complementary results such as UV, fluorescence, thermal denaturation, electrochemical and viscosity, and molecular docking studies under physiological conditions. Our results revealed that the Pemetrexed binds fairly strongly to dsDNA's minor groove through hydrogen bond interactions with the mostly adenine and guanine bases via its p-carbamide and p-carboxylic groups. MD simulations of the drug-dsDNA complex were followed for 50 ns to confirm that interaction is stable and robust electrostatic interactions were due to hydrogen bonding mostly with the adenine and guanine nucleotides in the minor groove. (c) 2021 Published by Elsevier B.V.