Introduction Congenital Adrenal Hyperplasia (CAH) is most often caused by mutation of the 21-hydroxylase gene (CYP21), which results in underproduction of cortisol with overproduction of precursor steroids and their metabolites by the adrenal glands. Historically the most common biomarker used for detecting CAH in pediatric patients is 17-Hydroxyprogesterone (17OHP). Another less commonly used biomarker for 21-Hydroxylase deficiency is 21-deoxycortisol (21DOF), which increases from very low levels in normal patients to high levels in affected patients as 17OHP rises to very high levels. In this study we performed retrospective analysis of serum 21DOF concentration in specimens that had been genotyped for mutations in the CYP21A2 gene, or had been submitted to our laboratory for provocative adrenocorticotropin (ACTH / Cosyntropin) stimulation testing. Methods: Biochemical testing for 21DOF concentration was measured by LC-MS/MS. Briefly, a TX-4 HPLC system (Thermo-Fisher) with Agilent® 1100 pumps (Agilent Technologies, Inc.) and a Sciex® 5000 (Danaher) triple quadrupole mass spectrometer in positive mode atmospheric pressure chemical ionization (APCI) was used for detection in Multiple Reaction Monitoring (MRM) mode. Genetic testing was performed using the CAHDetx test, which detects the 12 most common small mutations and large gene deletions/conversions in CYP21A2. Genetic Correlations: 21DOF was quantifiable (above the LLOQ of the assay) in 4% (n=24/600) of specimens where no mutation was detected. 21-DOF was quantifiable in 42% (122/292) of specimens with 21-hydroxylase enzyme mutations as determined by the CAHDetx test. Those mutations included In2G, I172N, V281L and others. Some mutations such as Q318X did not result in a detectable increase in 21-deoxycortisol. ACTH Stimulation Response: 21-deoxycortisol was below the quantitation limit in both the baseline and stimulated samples in ~35% (52/148) of submitted samples. The 21-deoxycortisol was quantifiable in only the stimulation sample in ~45% (65/148) of ACTH stimulation submitted, and was quantifiable in both baseline and stimulated samples in the remaining ~20% (30/148) of ACTH stimulation pairings. The extent of 21-deoxycortisol increase ranged from 1.2-fold to 116-fold with a median 14-fold increase. Clinical Significance: The use of 21-deoxycortisol may be beneficial in reducing the rate of false positives in CAH diagnosis when used in concert with other steroid hormones, and may eventually reduce the need for provocative testing to confirm CAH diagnosis.