In several counties, Vitex negundo have been claimed to have anti-inflammatory, analgesic, and antioxidant activities. It's frequently used in folk medicine for the treatment of various pain disorders. The methanol extract of Vitex negundo leaves was studied for its biological effects. To investigate different biological exertions of the methanol extract of Vitex negundo, the leaf extracts were obtained with pure methanol. In-vitro anti-oxidant activity was resolved using a DPPH radical scavenging assay. A clot lysis test was used to assess the thrombolytic activity. Antiproliferative effects of DMSO Vitex negundo in cell lines acquired from human cervix adenocarcinoma (HeLa cells). Using the brine shrimp lethality bioassay method, the plant extracts were evaluated for cytotoxic action. LC₅₀ values were determined through probit analysis of mortality percentages. In hole cross-test and open-field test, neuropharmacological activities of mice were assessed in mice. The findings revealed that Vitex negundo leaf extract possessed antioxidant effects, streptokinase exposed significant clot lysis and human cervix adenocarcinoma cells demonstrated mild cervical antiproliferative activity. Significant neuropharmacological action (antidepressant and anxiolytic) and cytotoxic action were shown by the methanol extract of Vitex negundo leaves.