In Ayurvedic system of medicine Decalepis hamiltonii has been used as traditional medicine to treat various disorders like cytotoxicity, oxidative stress, bacterial infections, inflammation. The aim of the current study was to determine the cytotoxicity, antioxidant potential of different fractions of D. hamiltonii including n-butanol, aqueous, chloroform. Among the fractions studied n-butanol, exhibited good antioxidant DPPH assay, superoxide Phenols and flavonoids were quantified and analysed using HPTLC. Declapeis hamiltonii cytotoxicity on Hep G2 cells. HPTLC detected bioactive substances. Comparing rf values of tested chemicals with standard values predicted flavonoids, glycosides. Flavonoid glycosides and those with RF values of 0.23 and 0.35 may exist.The peaks at 0.17, 0.67, 0.57, 0.74 and 0.82 steroid or triterpene or glycoside. Bioassay-guided extraction and fractionation was utilized to identify Hydromethanolic extract of D. hamiltonii root which demonstrated Hepatoprotective efficacy on CCl4-induced toxicity on Hep G2 cells. n- butanol fraction has the most protective effect. Percent cell viability was found to be 94.2 %.In addition, LC-MS/MS was used to analyze chemical characterization of bioactive molecules through bioassay guided approach. The result showed n-butanol fraction of D. hamiltonii extract was most promising regarding antioxidant, hepatoprotective activities because of presence of flavonoids specially isoflavones. Glucocaffeic acid compound showed an excellent docking score of 95.78, 125.78, 162.14 and 120.62 Kcal/mol with all four targeted enzymes and receptors TGF-beta, NF-kB, iNOS and COX-2 respectively. The results indicated n-butanol fraction exihibit protective effect activity against CCl4 induced toxicity in Hep G2 cell lines.