The Vitex negeundo is a widely used medicinal plant which has not been fully investigated in the past. We assessed the in vivo hepatoprotective and in vitro antioxidant, antibacterial, cytotoxicity and anti proliferative study of leaf extracts of V. neugundo. The chemically profiled using HPLC, three flavonoids were quantified and GC-MS analysis revealed the presence of two new compounds those were not reported earlier from the leaf extract of V. neugundo. The animal study was conducted on mice treated with CCl4 using methanolic and chloroform extracts (100, 200 and 300mg/kg b.w), with silymarin as a positive control. Hepatoprotective effects were determined by analyzing blood for liver marker enzymes, direct bilirubins and hematological parameters (RBC, WBC and platelets). The methanolic extract (300mg/kg b.w) has shown the stronger hepatoprotective effects against abnormalities produced by CCl4. The in vivo hepatoprotective effects correlated well with the in vitro antioxidant, cytotoxicity and antiproliferative activities and with high levels of flavonoids and other organic compounds analyzed from plant extracts. The leaf extracts of this plant could be good candidates for lead compound required for the development of antioxidant/anticancer drugs.