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Abstract: Background: Newborn screening for congenital adrenal hyperplasia (CAH) is commonly accomplished by measurement of 17-α-hydroxyprogestrone (17-OHP) using enzyme immunoassay (EIA). EIA contributes a significant number of false positives. Therefore, second-tier steroid profile by liquid chromatography–tandem mass spectrometry (LC–MS/MS) is warranted. Methods: Dried blood spots (DBS) were extracted with a mixture of methanol and water containing the deuterium labeled internal standards of d8-17-OHP, d7-androstenedione, and d4-cortisol. The final extracts were analyzed for 17-OHP, androstenedione and cortisol by LC-MS/MS in the multiple reaction monitoring (MRM) mode. Results: Mean recoveries of the target analytes, 17-OHP, androstenedione and cortisol, were between 97 and 115% with an average intra- and inter-assay CVs ranging from 3.9–9.9% to 3.6–10.1%, respectively. The high efficiency of this method enabled us to test 11,598 specimens, identified as indeterminate by EIA in ~6 years; resulting in 809 presumptive positives reducing the false positives rate by 93%. Conclusions: The three steroid profile provided better screening outcomes of CAH than 17-OHP concentration alone. Our sample preparation allowed high throughput using common laboratory chemicals. Using three internal standards significantly improved method precision and accuracy. The reduction in false positives significantly reduces anxiety for newborns and their families. [Copyright &y& Elsevier]