In a pursuit for effective anticancer hit/lead, a series of new β-carboline tethered quinoline-4-carboxamides, adjoining noticeable pharmacophoric properties, were synthesized. The in vitro cytotoxicity evaluation established that many of the compounds exhibits significant cytotoxicity (IC50 <25 μM) on various cancer cell lines like HCT116, SK-MEL-28, A549, and MCF-7. Precisely, 2-(4-chlorophenyl)-N-((1-(3-(trifluoromethyl)phenyl)-9H-pyrido[3,4-b]indol-3-yl)methyl) quinoline-4-carboxamide (compound 12e) was found to be most potent against breast cancer cell line (MCF-7) with an IC50 value of 5.71 μM. In addition, cytotoxicity evaluation against normal kidney cell line (HEK-293) revealed the cytospecificity and selectivity index of 12e. The compound 12e was found to induce apoptosis in MCF-7 evident by various traditional apoptosis assays advising morphological changes, nuclear alterations and generation of ROS. The flow cytometric analysis revealed significant early and slight late-stage induction of apoptosis. The target-based assays specify the ability of compound 12e to bind with DNA and enzyme assay revealed the inhibition of Topo II. Furthermore, molecular modeling studies uphold the significant DNA interactions of 12e with DNA base pairs along with promising drug-like properties for in silico prediction of physicochemical parameters.