Background: Ceramides are structural long-chain lipid molecules essential for cell architecture, with many biological functions involving cell proliferation, inflammation, apoptosis, and cell death. Ceramides have a diverse role in carcinogenesis depending on their chain length, metabolism, and localization. Disturbance in ceramide metabolism has been observed in many cancers such as head and neck, colorectal, lung, and liver. Objective: The current study aimed to use a high-performance liquid chromatography-tandem mass spectrometry analytical method for the measurement of C16 ceramide and C24 dihydroceramide in the serum of patients with hepatocellular carcinoma and chronic liver cirrhosis and to verify their potential role as markers of hepatocellular carcinoma. Patients and methods: This case control study enrolled 100 patients diagnosed with hepatocellular carcinoma and chronic liver cirrhosis (n= 50 each) and 50 healthy subjects as a control group. Serum samples were subjected to separation of ceramides by ultra-performance liquid chromatography and quantitative detection and measurement by tandem mass spectrometry. The assay method allowed accurate measurement of C16 ceramide and C24 dihydroceramide at a serum concentration <2.5 ng/ml. Results: The calibration curves' linearity had regression coefficients >0.98, with intra-day and inter-day precision and accuracy ranging from 89%-102%. C16 ceramide and C24 dihydroceramide were significantly higher in hepatocellular carcinoma and chronic liver cirrhosis patients than in healthy subjects (P<0.05). Their serum levels were not different in liver cirrhosis and hepatocellular carcinoma (P>0.05) but showed an increasing trend to be higher in hepatocellular carcinoma. Both ceramides did not correlate with the clinical parameters of hepatocellular injury as Child-Pugh score, BCLC stage, or the burden of tumor of all P>0.05. Conclusion: Quantitative measurement of C16 ceramide and C24 dihydroceramide by tandem mass spectrometry is an accurate and precise method for laboratory workup in patients with chronic liver cirrhosis.