Liver disease remains a major critical challenge in Thailand due to viral hepatitis. Clinical management requires close monitoring of liver fibrosis severity. Non-invasive testing is an attractive method for probing of disease progression. Mac-2 binding protein glycosylation isomer (M2BPGi) is a novel serum marker for fibrosis staging. The current study evaluates the marker among healthy donors and hepatitis C (HCV) patients. 100 HCV subjects were evaluated by liver biopsy. These patients had varying fibrosis severity based on METAVIR scores. Healthy donors were confirmed based on normal liver functions tests. Comparisons of M2BPGi levels among different study groups were performed and the effectiveness was evaluated using receiver operating characteristics (ROC) curves. Using liver biopsy as the reference standard, median M2BPGi levels in HCV cases were 0.74, 1.38 and 2.88 COI for F0-1, F2 and > F3 cases respectively. In healthy donors, the baseline values ranged 0.1-0.24 COI and statistically lower than liver disease cases profiled using M2BPGi. ROC analysis demonstrated superior results for M2BPGi levels among diseased populations and healthy controls. AUROC was determined at 0.983. Comparing with other non-invasive tests, M2BPGi showed a positive linear trend that indicated a strong match to existing methodologies. M2BPGi addresses a critical need in the management of liver disease by providing straightforward means to probe fibrosis severity. In this study, we found significant differences between hepatitis C and healthy subjects and established the background level in healthy donors.