<jats:title>Abstract</jats:title> <jats:p>Background: Congenital adrenal hyperplasia is a group of autosomal recessive disorders caused by a deficiency of 1 of 4 enzymes required for the synthesis of glucocorticoids, mineralocorticoids, and sex hormones. Analysis of 11-deoxycortisol (11DC), 17-hydroxyprogesterone (17OHP), 17-hydroxypregnenolone (17OHPr), and pregnenolone (Pr) in blood allows detection of these enzyme defects. <jats:p>Methods: The steroids were extracted from 200 μL of serum or plasma by solid-phase extraction, derivatized to form oximes, and extracted again with methyl t-butyl ether. Instrumental analysis was performed on an API 4000 tandem mass spectrometer with electrospray ionization in positive mode and multiple reaction-monitoring acquisition. <jats:p>Results: The limits of detection were 0.025 μg/L for 11DC, 17OHP, and Pr and 0.10 μg/L for 17OHPr. The method was linear to 100 μg/L for 11DC, 17OHP, and Pr, respectively, and to 40 μg/L for 17OHPr. Within- and between-run (total) imprecision (CVs) were &lt;7.1% and 11%, respectively. Reference intervals for children in Tanner stages 1 through 5 and adult males and females for 17OHP, 11DC, Pr, and 17OHPr were established. Prepared samples were stable for &gt;72 h. <jats:p>Conclusions: The detection limit and selectivity of this method and its small sample volume requirement allow analysis of endogenous concentrations of adrenal steroids in serum or plasma from children and adults. The method thus has an important potential role in the evaluation of the status of 4 of the enzymes involved in adrenal steroid biosynthesis.