Direct immersion SPME-GC-MS-MS was used for the analysis of steroids in water at part-per-trillion(ppt) and lower concentrations. The method was validated and extended to real sample analysis. The method were linear from 0.01 to 5 ng/ml with precision less than 10% relative standard deviation for a steroid mixture at 1 ng/ml. Limit of quantitation and limit of detection was found to be 200- 1200 pg/L and 30-200 pg/L respectively and recoveries ranged from 88-103 %. To understand the extraction efficiency of the fiber, a depletion study was performed. The fiber/ sample partition coefficients for the steroids were determined to be 1.0 x 104 to 1.5 x 104 . The extraction was performed without derivatization or the use of an internal standard. SPMEGC-MS-MS effectively demonstrated ultra-trace level detection of steroids in water.Universidade Estadual Paulista Júlio de Mesquita Filho, Instituto de Química de Araraquara, Araraquara, Rua Professor Francisco Degni, Departamento de Química Analítica, Jardim Quitandinha, CEP 14800060, SP, BrasilUniversidade Estadual Paulista Júlio de Mesquita Filho, Instituto de Química de Araraquara, Araraquara, Rua Professor Francisco Degni, Departamento de Química Analítica, Jardim Quitandinha, CEP 14800060, SP, Brasil