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Background: An accurate measurement of testosterone is needed in many clinical applications for correct diagnosis and appropriate treatment. Our aim was to develop a fast and robust high-throughput LC-MSMS method for quantification of serum testosterone in women., Methods: Testosterone was derivatized by oximation and extracted with methyl tert-butyl ether from 200 microL of serum. Further matrix elimination was achieved on-line using a column-switching LC-method. The instrumental analysis was performed on an API4000 tandem mass spectrometer equipped with an Agilent series 1312A binary pump and an Agilent series 1311A quaternary pump. The MRM transitions were 304-->124 and 304-->112 for testosterone and 307-->124 and 307-->112 for d(3)-testosterone., Results: The total analysis time of the column-switching method was 3 min. Linear calibration curves were obtained in the concentration range from 0.035 nmol/L (0.01 microg/L) to 6.92 nmol/L (2 microg/L). Within-day and between-day precision, expressed as the relative standard deviation at four different concentrations ranged from 4.70% to 9.35%. Correlation with the in-house method (solvent-extraction RIA) showed r(2)=0.920., Conclusions: The presented column-switching method offers a simple, fast and economical analysis of testosterone in human serum. The procedure requires only small sample volumes and is well suited for quantification of testosterone in serum from women and children.