Prolonged icterus is an important and common problem in neonatology and accurate determination of the different bilirubin species, including differentiation between delta-bilirubin and mono- and di-conjugated bilirubin, is useful for diagnostic purposes. However, most bilirubin measurements routinely performed in the clinical laboratory are hampered by the lack of separation of the four bilirubin fractions (unconjugated bilirubin, mono-conjugated bilirubin, di-conjugated bilirubin, and delta-bilirubin). Herein, we propose a high-performance liquid chromatography-based method, independent of commercially available standards or reliable molar absorption coefficients, for the determination of bilirubin fractions in blood samples from icteric patients. The method is a robust and reliable candidate for a semi-automatized setup for measuring the various bilirubin fractions in a specialized laboratory handling samples from clinics with expertise in biliary disease.