Congenital adrenal hyperplasia (CAH) is a rare, inherited disorder of adrenal steroid synthesis. In many countries it is part of the neonatal screening program enabling early diagnosis and treatment. In case of an abnormal neonatal screening result or when other differences of sexual development (DSD) are suspected, measurement of serum steroid hormones using liquid chromatography coupled to mass spectrometry (LC-MS/MS) is needed for further diagnosis. However, reliable age- and sex-specific reference intervals (RIs) for serum steroid hormones during the neonatal period are missing. We therefore aimed to establish LC-MS/MS based RIs for serum steroid hormones in neonates. Serum was obtained from healthy term neonates at two time points: 130 samples at day 3-8 (T1, time of the neonatal screening) and 126 samples at day 13-15 (T2,two weeks old). Concentrations of cortisol, cortisone, corticosterone, 11-deoxycortisol, 21-deoxycortisol, 11-deoxycorticosterone, testosterone, androstenedione, and 17-hydroxyprogesterone (17-OHP) were measured using LC-MS/MS. RIs (in nmol/L) were established for T1 and T2: cortisone (19.3-215;18.0-212), cortisol (10.0-407;8.4-446), corticosterone (<31;<50), 11-deoxycortisol (0.73-4.6;0.70-3.6), 17-OHP (<4.9;<5.1), androstenedione (0.3-1.8;0.3-2.7), 11-deoxycorticosterone (<0.2;<0.2), and 21-deoxycortisol (<1;<1), respectively. Testosterone differed between boys and girls: RIs at T1 and T2 for boys were 0.27-4.3 and 0.63-13.9, and for girls<0.30 and <0.47, respectively. We established LC-MS/MS based RIs for cortisol, cortisone, corticosterone, 11-deoxycortisol, 21-deoxycortisol, 11-deoxycorticosterone, testosterone, androstenedione, and 17-OHP in neonates in the first and second week of life.