Purpose Clinical application of biomarker candidates discovered by proteomic analysis is challenging. The purpose of this study was to standardize preanalytical conditions for measurement of serum levels of fibrinogen alpha C-chain 5.9 kDa fragment (FIC 5.9) and to test the diagnostic value of this peptide for detection of early hepatic fibrosis in patients with hepatitis C virus (HCV)-related chronic hepatitis. Experimental design Serum FIC 5.9 levels were measured by a sandwich ELISA. Effects on the serum FIC 5.9 level of temperature, the time between venipuncture and serum separation, and the types of collection tubes used were examined. The diagnostic value of serum FIC 5.9 as an early indicator of hepatic fibrosis due to HCV was then assessed. Results FIC 5.9 was produced in a time- and temperature-dependent manner after venipuncture. Abnormal FIC 5.9 values were found in 89.5% of FI stage patients. Receiver operating characteristic analyses confirmed the superiority of FIC 5.9 over other conventional markers for early detection of fibrosis. Conclusions and clinical relevance The serum FIC 5.9 level may be an early indicator of hepatic fibrosis in HCV-related chronic liver diseases. This study provides an example of a pipeline from biomarker discovery by proteome analysis to assay optimization and preliminary clinical validation.