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Quantitation of endogenous steroids and their precursors is essential for diagnosis of a wide range of endocrine disorders. Usually, these analyses have been carried out using immunoassays. However, immunoassays often overestimate concentrations due to assay interference by other endogenous steroids, especially for low concentrations. Mass spectrometry based methods offer superior specificity, accuracy, and sensitivity. We therefore present a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method with automated sample preparation for determination of 17α-hydroxyprogesterone (17OHP), cortisol, cortisone, dehydroepiandrosterone sulfate (DHEAS), androstenedione (A4), testosterone (T), and estrone sulfate (E1S). Samples were prepared using protein precipitation and 96-well filter plates, fully automated in a pipetting robot and analyzed by LC-MS/MS. Serum samples from 187 healthy children and adolescents aged 5-18 years were used to study hormone changes in relation to sex and pubertal stage. Lower limit of quantification for 17OHP was 0.7 nmol/L, for cortisol 11 nmol/L, for cortisone 2 nmol/L, for DHEAS 0.1 µmol/L, and for A4, T, and E1S, 0.2 nmol/L. This study showed a general increase in 17OHP, DHEAS, A4, T and E1S in both genders during puberty. In boys, A4 and T increased significantly throughout pubertal development. Girls had significantly higher A4 and E1S concentrations, while boys had higher T concentrations. No sex- or puberty-specific differences were seen in cortisol or cortisone concentrations. To the best of our knowledge, this is the first presentation of changes in serum E1S concentrations during pubertal development in healthy children. [ABSTRACT FROM AUTHOR]