The Dutch neonatal screening scheme for Congenital Hypothyroidism (CH) is primarily based on the determination of thyroxine (T4) in filter paper blood spots. In the lowest 5% of T4 values, thyroxine binding globulin (TBG) is measured in order to be able to correct for occasional low TBG levels. However, because the commercial TBG kit has been withdrawn from the market, alternative strategies are needed to be explored including the assessment of free T4.We evaluated the Neonatal Free Thyroxine (fT4) enzyme immunoassay (EIA) kit of Bio-Rad.FT4 as measured in a daily run of random samples correlated with T4. We also observed a correlation between fT4 and T4, and between fT4 and T4/TBG ratio in blood spots with low T4 concentrations. The correlation between fT4 and T4 in blood spots of proven CH-patients was highly significant. ROC curves were constructed for the fT4 assay and the T4/TBG ratio based on 27 CH patients and 215 controls with a complete set of data. The curves of both assays seemed to be rather similar.We conclude that the validity of the fT4 and the T4/TBG-approach seems to be the same. A study with a larger sample size giving the same or even more favorable results for the fT4-approach is necessary before we will change the present CH protocol. •The paper describes the evaluation of a free T4 enzyme immunoassay in blood spots.•The aim is to replace total T4 and TBG for a single fT4 measurement.•Despite a correlation between fT4 and T4 in CH-patients, no cut-off value can be established.•Determination of fT4 levels in blood spots was inferior to the present CH-protocol. © 2013 Elsevier B.V.