Proteomic profiling of serum is an emerging technique to identify new biomarkers indicative of disease severity and progression. The objective of our study was to assess the use of surfaceenhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) to identify multiple serum protein biomarkers for detection of liver disease progression to hepatocellular carcinoma (HCC). A cohort of 170 serum samples obtained from subjects in the United States with no liver disease (n ؍ 39), liver diseases not associated with cirrhosis (n ؍ 36), cirrhosis (n ؍ 38), or HCC (n ؍ 57) were applied to metal affinity protein chips for protein profiling by SELDI-TOF MS. Across the four test groups, 38 differentially expressed proteins were used to generate multiple decision classification trees to distinguish the known disease states. Analysis of a subset of samples with only hepatitis C virus (HCV)-related disease was emphasized. The serum protein profiles of control patients were readily distinguished from each HCV-associated disease state. Two-way comparisons of chronic hepatitis C, HCV cirrhosis, or HCV-HCC versus healthy had a sensitivity/specificity range of 74% to 95%. For distinguishing chronic HCV from HCV-HCC, a sensitivity of 61% and a specificity of 76% were obtained. However, when the values of known serum markers ␣ fetoprotein, des-gamma carboxyprothrombin, and GP73 were combined with the SELDI peak values, the sensitivity and specifity improved to 75% and 92%, respectively. In conclusion, SELDI-TOF MS serum profiling is able to distinguish HCC from liver disease before cirrhosis as well as cirrhosis, especially in patients with HCV infection compared with other etiologies. (HEPATOLOGY 2005;41:634-642.) T he incidence of hepatocellular carcinoma (HCC) continues to increase in the United States, 1 while, unfortunately, patient survival with HCC has only marginally improved over the last 20 years. Between 1981 and 1998, the 5-year survival rate only rose from 2% to 5%. 2 The poor survival rate is in part related to the diagnosis of HCC at advanced stages, where effective therapies are lacking. 3 Surveillance of patients at the highest risk for developing HCC (i.e., patients with cirrhosis) is an important strategy that can potentially decrease the cancer-related mortality rate. Although HCC meets the criteria of a tumor that would benefit from a surveillance program, the poor sensitivity and specificity of currently available tools has prevented widespread implementation of HCC surveillance. For example, ␣ fetoprotein (AFP) has been the serum marker that is most widely used for diagnosis as well as surveillance of HCC. 4,5 However, AFP levels may be normal in up to 40% of patients with HCC, particularly during the early stages (low sensitivity). 6 Furthermore, elevated AFP levels may be seen in patients with cirrhosis or exacerbations of chronic hepatitis (low specificity). 7 Prospective studies evaluating the performance characteristics of AFP for HCC surveillance