21-hydroxylase deficiency (21OHD) has a high prevalence of asymptomatic heterozygote carriers in the general population. The carrier status is important for genetic counseling in 21OHD. Heterozygosity is also associated with symptoms like hyperandrogenism. Moreover, there is a lack of a distinctive hormonal parameter for identifying carriers in the era of mass spectrometry. In this study we compared basal and ACTH-stimulated steroid hormones of heterozygous carriers of CYP21A2 mutations and of control individuals in order to establish a distinctive parameter for separation of heterozygote carriers from normal controls. We measured basal and ACTH-stimulated concentrations of 11 steroid hormones using LC-MS/MS. The study included 58 heterozygote carriers of CYP21A2 gene mutations (35 males, 23 females, age range 6 – 78 years) and 44 independent control subjects (25 males, 19 females, age range 8 – 58 years). All participants were allocated either to the group of the heterozygous carriers or the healthy controls based on CYP21A2 genetic analysis. Heterozygotes could be identified best applying the [(17OHP+21S)/F*1000] ratio 30 min after ACTH injection. An optimal cut-off value of 8.4 provided 89% sensitivity and specificity. Considering this data and a published frequency of heterozygotes with classic mutations of 1/50 to 1/61, the positive predictive value of this cut-off is only 12%. However, the negative predictive value excluding heterozygosity in a given patient is 99.8%. We conclude that considering the only marginal biochemical effects anticipated from heterozygous CYP21A2 mutations, the stimulated [(17OHP+21S)/F*1000] identifies and excludes heterozygotes remarkably well. Never-the less, LC-MS/MS cannot replace genetic testing since the sensitivity and specificity did not reach 100% applying the optimal cut-off of 8.4. However, due to the considerably high negative predictive value of the optimal cut-off and due to a specificity of even 100% applying a cut-off higher than 14.7, hormonal assessment of heterozygosity can be of significant aid in conditions with limited access to genetic testing. Moreover, the [(17OHP+21S)/F*1000] ratio can guide further diagnostic considerations in the differential diagnosis of hyperandrogenism.