Objective: In this study, we determined the assay performance criteria necessary to produce acceptable results for ≥ 98% of neonate bilirubin samples collected by capillary heel-stick. Study design and methods: We determined serum free hemoglobin levels in 151 heel-stick serum samples to determine the hemolysis level. We then tested the effect of hemolysis on total bilirubin levels determined by four commercially available assays. Results: The mean level of serum free hemoglobin was 1.62 g/L. Of the serum total bilirubin assays tested, the Total Bilirubin Special (Roche Diagnostics) and the TBILI (Roche Diagnostics) reagents did not show significant interference at the concentrations of free hemoglobin observed in ≥ 98% of heel-stick samples. The Vitros Bu/Bc slide (Ortho-Clinical Diagnostics) showed significant interference only at normal bilirubin concentrations; while the Bilirubin DPD reagent (Amresco Inc.) showed significant interference starting at hemoglobin concentrations of 1.0 g/L. Conclusions: Bilirubin assays that are not sensitive to ∼ 6 g/L free hemoglobin should provide accurate results for most samples obtained via capillary heel-stick. Of the four assays tested, the Bilirubin DPD reagent (Amresco Inc.) was the most susceptible to the presence of free hemoglobin and will result in a higher rejection rate of neonate capillary heel-stick samples.