Recently, a new signaling complex Death Associated Protein Kinase 1 (DAPK1) ̶ N-methyl-D-aspartate receptor subtype 2B (NMDAR2B or NR2B) engaged in the neuronal death cascade was identified and it was found that after stroke injury, N-methyl-D-aspartate glutamate (NMDA) receptors interact with DAPK1 through NR2B subunit and lead to excitotoxicity via over-activation of NMDA receptors. An acute brain injury, such as stroke, is a serious life-threatening medical condition which occurs due to poor blood supply to the brain and further leads to neuronal cell death. During a stroke, activated DAPK1 migrates towards the extra-synaptic site and binds to NR2B subunit of NMDA receptor. It is this DAPK1-NR2B interaction that arbitrates the pathological processes like apoptosis, necrosis, and autophagy of neuronal cells observed in stroke injury, hence we aimed to inhibit this vital interaction to prevent neuronal damage. In the present study, using PubChem database, we applied an integrative approach of virtual screening and molecular dynamic simulations and identified a potential lead compound 11 that interrupts DAPK1-NR2B interaction by competing with both ATP and substrate for their binding sites on DAPK1. This inhibitor was found potent and considerably selective to DAPK1 as it made direct contact with the ATP binding sites as well as substrate recognition motifs: Gly-Glu-Leu (GEL) and Pro-Glu-Asn (PEN). Further in vitro and in vivo experiments are demanded to validate the efficacy of compound 11 nevertheless, it can be considered as suitable starting point for designing DAPK1 inhibitors. © 2017 Elsevier Inc.