Background: Barleria lupulina Lindl. (Hop-headed) is a small shrub, possess potent anti-inflammatory, analgesic, anti-leukemic, antitumor, anti-hyperglycemic, anti-amoebic, virucidal, diuretic, bactericidal and antibiotic properties. Methods: Cytotoxicity, bioactive assay and genetic analysis of B. lupulina were investigated in the present communication. The leaf extract was investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), Neutral red uptake (NRU), DNA fragment, reactive oxygen species (ROS) generation, mitochondrial membrane potential (MMP) assay, gene expression analysis and cDNA synthesis to evaluate anti-cancerous potency using cancerous THP-1 cell lines in vitro and in vivo. Results: HPTLC analysis reveals four spots and GC-MS analysis displayed the presence of eleven bioactive compounds among which benzofuranon, hexadecanoic acid, ethyl 9,12,15-octadecatrienoate, and 3,7,11,15-tetramethyl-2-hexadecanoic acid were the most prominent compounds. The ethanolic extract showed significant cytotoxicity (P<0.5) against THP-1 cell line at a concentration of 1mg/mL. The cells were also observed for apoptosis through DNA fragmentation in B. lupulina treated cells. Conclusions: It can be concluded that if the dose range was further refined within the range of 100-1000 µg/mL there could be dose at which the entire population of the THP-1 cell line would be apoptosis induced. The extract induced ROS in the cells after 30 minutes of exposure displaying cytotoxic effects and DNA fragmentation assay.