Diagnosis and management of infants and children with sex steroid disorders require fast and simultaneous assessment of several sex steroid metabolites in serum at low concentrations and on small sample volumes. Therefore, we developed a sensitive and selective TurboFlow-LC-MS/MS method for quantification of DHEA, DHEAS, 17α-hydroxyprogesterone, Δ4-androstenedione and testosterone in serum from pre-pubertal children. Run time was 10.75min. Limits of quantification were as follows: DHEA, 0.88nM; DHEAS, 48nM; 17α-hydroxyprogesterone, 0.19nM; Δ4-androstenedione, 0.18nM and testosterone, 0.10nM. Intra-day relative standard deviation ranged from 4.6 to 13.8% and inter-day relative standard deviation ranged from 5.7 to 15.7%. Steroid concentrations in 38 serum samples from pre-pubertal children were compared with results obtained by immunoassays for DHEAS, Δ4-androstenedione and testosterone. DHEAS gave overall similar results but with several outliers, while levels of Δ4-androstenedione were found to be much lower when analysed by LC-MS/MS. Testosterone was not detected in any of the samples analysed using a sensitive immunoassay, while 30 of 38 samples were quantifiable using the current LC-MS/MS method. The presented method is suitable in a clinical setting for simultaneous quantification of five steroids important for management of children with disorders of sex development and steroid biosynthesis defects.