Background: Carcinoma of the liver is the sixth most frequent malignancy in humans and is accountable for more than 600,000 deaths annually. According to the statistics, hepatocellular carcinoma patients typically die within a year after being diagnosed. As 5-fluorouracil (5-FU), the primary chemotherapeutic agent for most different types of cancers, is now routinely monitored via therapeutic drug monitoring, it has contributed to better clinical results. Many studies indicated the role of nitric oxide (NO) in different cancers as a pro-neoplastic or anti-neoplastic effector, but its function remains unclear in hepatocellular carcinoma (HCC). Aim of the study: The study's overall purpose was to look into the efficacy and toxicities of 5- fluorouracil on the biological behavior of HepG2 cell line by the methyl thiazole tetrazolium (MTT) assay and evaluate nitric oxide levels in monitoring the progression of HCC. Material and Methods: HepG2 cell line was incubated with different concentrations of 5-Fluorouracil as a chemotherapy drug. The effects of 5-Fluorouracil on cell proliferation, morphology, and the half-maximal inhibitory concentration (IC50) values were determined by the methyl thiazole tetrazolium (MTT). Results: The IC50 values for 5-Fluorouracil in HepG2 cells was (12.92±0.085μg), moreover the NO levels in the HepG2 cell line treated with 5-Fluorouracil were measured. We found significant inhibition in NO levels after treating HepG 2 cell lines by 5-Fluorouracil. Conclusion: The change in NO levels following chemotherapy helps predict treatment response in HepG-2 cells.