Hepatocellular carcinoma (HC) is often difficult to distinguish from secondary liver neoplasia (SLN) by physical and imaging diagnostic procedures alone. To this aim we have extended and improved a laboratory approach based on a serum lactate dehydrogenase isoenzyme ratio (LD4:LD5) by adding the carcinoembryonic antigen: alpha-fetoprotein ratio, alkaline phosphatase, and serum iron concentrations to obtain a highly efficient discriminant function. In two successive cohorts, for a total of 102 patients, all histologically diagnosed, with a prevalence of HC vs SLN of 3:1, we correctly classified 96% of cases (100% of SLN cases). Subsequent verification with the jackknife reallocation statistical algorithm confirmed these results. In conclusion, this discriminant function based on simple laboratory assays of a few analytes is an important tool in solving a diagnostic dilemma in cases of liver neoplasia.