An isotope-dilution TurboFlow-LC-MS/MS method for simultaneous quantification of the ten steroid metabolites dehydroepiandrosterone sulfate (DHEAS), progesterone, 17α-hydroxyprogesterone (17-OHP), Δ4-androstenedione (Adione), corticosterone, 11-deoxycortisol, cortisol, cortisone, testosterone (T), and estrone 3-sulfate (E1-S) in serum was developed and validated. Limits of quantification, variability (inter- and intra-day), analytical range and linearity were all found to be acceptable for clinical use. Furthermore, sample stability was evaluated including the influence of freeze-thaw cycles and the effects of temperature and storage time. The method was applied to 391 serum samples from healthy, Danish boys 10–18 years old. The concentration ranges of the included steroid metabolites for this population are presented. Concentrations of DHEAS, 17-OHP, Adione and T in the 391 serum samples were furthermore compared to results obtained using an existing LC-MS/MS method in our laboratory. Excellent agreement was found between the methods. Furthermore, the improved sensitivity of the new method allowed for quantification of a number of samples found to be below the LOQs of the existing method. Thus, the two instruments and their associated methods were validated as possible back-ups for each other, which we consider an extremely important issue in high-throughput laboratories analyzing clinical samples on a regular basis. The ten analytes included can be analyzed simultaneously but it is also possible only to include some of these analytes for specific diagnostic purposes which make the new method an extremely useful tool in the clinical laboratory.