The hepatitis B virus X protein (HBxAg) is responsible for severe complications of HBV infections including primary hepatocellular carcinoma. A sandwich type ELISA and a flow cytometric microbead assay for quantitative determination of serum levels of Hbx-Ag are introduced. We have previously developed monoclonal antibody families against well-conserved epitopes on HbxAg, characterized by different immunohistochemical and immunoserological techniques. Special selection of the antibody pairs provided highly sensitive and highly specific tools for quantitative immunoassay development. The resulting assays were tested on human sera (208 samples) collected from patients suffering from different clinical forms of HBV infection. The sensitivity range of the sandwich type ELISA was between 4 and 2000 ng/ml as measured on both the recombinant antigen and the sera of chronic hepatitis patients. A further flow cytometric microbead assay was established and tested in parallel with the ELISA. The quantitative results of these two immunoserological techniques were in strong correlation and they were found to be highly specific and sensitive on clinical samples. The HBxAg ELISA technique is applicable for routine clinical laboratory measurements, and our HBxAg microbead technique is recommended for complex multiparametric measurements combined with other markers.