Solid-phase microextraction combined with fast short-column liquid chromatography/mass spectrometry (SPME/LC/MS) was used for isolating and analysing 11 corticosteroids and 2 steroid conjugates from urine samples. Several SPME parameters such as polarity of fibres, extraction time and effect of ionic strength, were investigated, and their impact on the SPME/LC/MS technique was studied. To demonstrate the potential of the SPME/LC/MS method, its application to the determination of steroids in human urine was investigated. The method was shown to be sensitive with detection limits between 4 and 30 ng/mL and precision between 4.9 and 11.1% RSD through the use of an internal standard technique. The versatility of the method was also exhibited by its excellent linearity in the concentration range of 20 to 20,000 ng/mL in urine for all investigated compounds. A comparison of the SPME/LC/MS method with LC/MS methods utilizing traditional sample preparation techniques, shows that the former offers similar performance in terms of precision, linearity and detection limits, but is clearly easier to use and faster to perform.