Background: Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death worldwide nowadays. In Egypt, the cancer is frequently discovered at an advanced stage, at which no therapy, even surgery, is successful. Early detection of the disease is critical because it enables the patients to be treated prior to enlarging or metastasizing to distant organs. A tumor marker is a serological agent whose blood level may indicate the existence of the tumor in the early stages. The gold standard and more reliable biomarkers for HCC is alpha-fetoprotein (AFP). Aim of the study: To determine the cytotoxicity and Selectivity index of 5-Fluorouracil in HepG2 and WI-38 cell lines by the methyl thiazole tetrazolium (MTT) assay and assess alpha-fetoprotein (AFP) levels as a tumor marker in the progression of HCC. Material and Methods: HepG2 and WI-38 cells were incubated with different concentrations of 5-Fluorouracil as a standard chemotherapy drug. The half-maximal inhibitory concentration (IC50) values were determined the methyl thiazole tetrazolium (MTT) for 5-Fluorouracil. Results: IC50 values for HepG2 and WI-38 were found for 5-Fluorouracil (32.533±0.777μM and 63.400±0.624μM ), respectively. Moreover, the selective index value of 5-Fluorouracil was (1.949±0.027μM). The AFP levels in the HepG2 cell line treated with 5-Fluorouracil were measured. We found significant inhibition in AFP levels after treating HepG 2 cell lines by 5-Fluorouracil. Conclusion: The change in AFP levels following chemotherapy with 5-fluorouracil is beneficial for predicting treatment response in the HepG-2 cell line.