Phytocannabinoids and synthetic cannabinoids have been explored as the lead due to their anti-proliferative nature and can be anti-cancer agents. These were found to activate numerous pharmacological targets to generate new therapies in alleviating specic symptoms or delaying the disease of Cancer. However, the comprehensive anti-cancer activities of cannabinoid acids and non-cannabinoids are not fully explored. Herein we report simple extraction, faster bioassay-guided fractionation, and HPLC-assisted purication of bioactive secondary metabolite and their identication. Anti-proliferative activity of six cannabinoid acids (CBDA’s) and eleven non-cannabinoids from the leaves of Cannabis sativa was evaluated. HPLC- assisted purication from the bioactive fractions leads to isolate one new compound Methoxy Canniprene (methoxy isoprenyl bibenzyl), including all six CBDA’s eleven non-cannabinoids in a single step, and NMR, HR-ESI-MS studies, and comparison with the literature data authenticated their structures. The crude extract (IC50 range: 18.0–37.2 μg/mL), Fraction A to D (11.9 to 222 μg/mL), and puried different cannabinoid and non cannabinoids (IC50 range: 0.5–100 μM) displayed potent anti-proliferative activities against almost all the tested ve human cancer cell lines.