Summary: Loss of hepatitis 'e' antigen (HBeAg) in patients with HBeAg-positive chronic hepatitis B is associated with improved long-term clinical outcome and is defined as a goal of antiviral treatment by clinical practice guidelines. Recent studies suggest that baseline levels and on-treatment monitoring of HBeAg levels may identify patients most likely to respond to therapy. The aim of this study was the development of a protocol for the quantitative determination of HBeAg using the Elecsys(�) HBeAg immunoassay. The linear range of the Elecsys(�) HBeAg immunoassay was established using recombinant HBeAg and five different diluents. The assay was validated against the Paul Ehrlich Institute (PEI) international standard serum. Linearity was demonstrated up to a cut-off index (COI) of 1000, independent of the diluent used. Optimal linearity was obtained using the Elecsys(�) Universal Diluent. Using the PEI reference standard, conversion factors were established as 4.50 COI for 1 PEIU/mL corresponding to 0.222 PEIU/mL for a COI of 1. Based on the results from these analyses, a simple algorithm for the quantitative measurement of HBeAg using the Elecsys(�) HBeAg immunoassay was developed. Using a simple algorithm with an initial 1:40 dilution, the Elecsys(�) HBeAg assay provides robust quantification of serum HBeAg in an easy-to-use and rapid system. The use of a commercially available, standardized diluent improves comparability between laboratories.