Background Diagnosis of Congenital Adrenal Hyperplasia (CAH) is based on the quantification of 17-hydroxyprogesterone (17-OHP), usually by immunoassay. During the neonatal period the specificity of screening for CAH by blood spot 17-OHP immunoassay is low. High false-positive rates result in a relatively high demand for a second-tier serum confirmation test. A robust, specific and selective method for measurement of cortisol, 21-deoxycortisol, 11-deoxycortisol, 4-androstene-3,17-dione (A4) and 17-OHP in serum has been developed. The method involves a simple extraction procedure and a fast analysis using ultra-performance liquid chromatography–tandem mass spectrometry (UPLC/MS/MS). Methods The steroids were extracted from 50 µl of serum using methyl-tert-butyl-ether. Analysis was performed on a UPLC tandem quadrupole mass spectrometer system in positive mode electrospray ionization and multiple reaction monitoring acquisition. Results The assay was linear over each analyte concentration range with all correlation coefficients (r2) > 0.996. Inter- and intra-day CVs were ≤ 10% across the analytical range. In addition simultaneous measurement of the full range of steroids on the pathway to cortisol allows confirmation of the affected steroidogenic enzyme. Conclusions A second-tier test for the confirmation of CAH has been developed. The method allows for detection and quantification of 5 steroids related to CAH over the range of the clinical assay with good linearity, sensitivity and precision.