¹Department of Pharmaceutical Technology, Jadavpur University, Kolkata-32, West Bengal, India DOI : 10.29228/jrp.693 The assessment of bioactive ingredients with potential hepatoprotective properties focuses primarily on in-vitro bioassays. This study aimed to assess the effectiveness of pre-treating primary hepatocyte cells with hepatoprotective earthworm extract to prevent damage from carbon tetrachloride (CCl4). Cell viability, aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lipid peroxidation or malondialdehyde (MDA) levels activities, as well as hepatoprotective activity of Earthworm extract protein powder (EEP) different concentration were 25, 50, and 100 μg/ml, Goat milk powder (GP) and Silymarin (SLM 100 ug/ml), and CCl4 were assessed in-vitro assay on the laboratory model rat primary hepatocyte culture. The selection of the CCl4 minimum lethal dosage might occasionally make examining liver damage more time consuming. Therefore, we examine CCl4 effectiveness at low to high doses in primary rat hepatocytes. Using primary hepatocyte as a toxicity model, we explored the cellular toxicity and oxidative stress related to CCl4 exposure. Our findings imply that the CCl4-induced cytotoxicity is fundamentally influenced by oxidative stress. The MTT assay results exhibited that CCl4 lower to higher doses decreased the viability of primary hepatocytes within six hours of treatment and hepatoprotective activity of EEP was identified. The lipid peroxidation assay revealed that MDA levels were inhibited by EEP and GP treatment in primary hepatocytes. Our results also showed EEP and GP treatment significantly inhibited AST, ALT, inflammatory marker Tnf-α and MDA level increase due to inducing CCl4 in in-vitro rat primary hepatocytes. According to the studies, EEP and GP function as an efficient hepatoprotective agent and prevent CCl4 treatment-induced primary hepatocyte toxicity. Keywords : Hepatoprotective; Primary hepatocyte; Earthworm extract protein; Goatmilk; Silymarin