Background: The simultaneous quantification of a steroid hormones panel provides more clinical information than a single steroid assay. Traditionally, steroids have been quantified with immunoassays which are characterized by high rate of positive results. Aim of this work, was to develop a TurboFlow-LC-MS/MS method for the simultaneous quantification of 17-hydroxyprogesterone, androstenedione, cortisol and testosterone in serum. Methods: To 100 mu L of serum, 100 mu L of internal standard solution in methanol were added; after centrifugation the supernatant was injected in the TurboFlow for further purification. Steroids were determined using a TSQ Vantage operating with an atmospheric pressure chemical ionization source. Method was fully validated and results compared with immunoassay methods. Results: Limit of quantification ranged from 0.02 ng/mL to 1 ng/mL. The precision was lower than 11% and accuracy ranged from 93.5 to 121.6%. The correlation was acceptable for all analytes except for low levels of testosterone. However, the Bland-Altman plots display a positive bias for androstenedione and 17-hydroxyprogesterone, and a negative bias for cortisol and testosterone. Conclusions: TurboFlow analysis provides a simple and effective clean-up procedure minimizing the interference of the matrix. The presented method is selective, precise, and sensitive being suitable in a clinical laboratory.