AbstractSignificanceBilirubin forms by the breakdown of heme proteins in the liver, but a newborn’s sluggish liver can lead to elevated serum bilirubin levels that cross the blood–brain barrier and result in kernicterus. Earlier studies have used the 400 to 500 nm optical wavelength range to characterize the bilirubin content. There is not a universally established correlation among other wavelengths and the amount of bilirubin in clinical whole blood samples.AimWe demonstrated that the amount of bilirubin could be quantified with∼82%accuracy in a label-free, self-referenced manner using only a few wavelengths, viz. 468, 492, 500, 560, 605, 645, 660, and 675 nm, wherein band-averaged absorption measurements are used.ApproachWe addressed the above problem by conducting a preliminary study containing 50 neonates through an absorption spectrum measurement of whole blood in 3 to5μlsamples from the neonates. We constructed a hierarchical decision method that first grossly divides the 30 neonates of the training set into<10mg/dland≥10mg/dlbilirubin level cohorts. A subsequent boundary condition further divides the≥10mg/dlgroup into two>15mg/dland≤15mg/dlbilirubin level cohorts. A finer measure later predicted the bilirubin content of each of these groups as low (<10mg/dl), medium (10 to15mg/dl), and high (>15mg/dl).ResultsUsing this hierarchical decision model statistical approach, we quantified the amount of bilirubin in the 20 testing set samples with 82% accuracy.ConclusionsWe formulated a biostatistical model in which we automated the spectrometric determination of total bilirubin in the whole blood for patients of neonatal hyperbilirubinemia.