The separation of the three major isomers and the main photoproducts of bilirubin by Ion-Pair High Pressure Liquid Chromatography is described. Chromatographic procedures found in the literature proved difficult to reproduce, had unacceptably long elution times (over 2 hours), required complex gradients or two or more columns. As these reports provided no data to show why specific chromatographic conditions were chosen an optimization study was carried out. The separation is shown to be dependent on pH, organic content and buffer concentration of the mobile phase, ion-pairing agent concentration and injection solvent. A separation, carried out in less than thirty minutes on octadecyl silica using isocratic conditions with detection at 455 nm is demonstrated. This technique will be used in the elucidation of the mechanism involved in phototherapy treatment of neonatal jaundice.