The solid-phase micro-extraction (SPME), Bis(trimethylsilyl) trifluoroacetamide (BSTFA) headspace derivatization on the SPME fiber, and gas chromatography–mass spectrometry (GC/MS) analysis of steroids from aqueous and biological solutions are shown. The effects of extraction and derivatization parameters have been shown. The extraction time of several steroids was shown to be approximately 30 min., which is typical of SPME methods. The analyte solution pH and ionic strength may have dramatic effects on the extraction recovery and should be optimized as part of method development. The incubation temperature may have several influences on the recovery from the derivatization reaction. Each analyte has a separate optimum temperature for derivatization. Thus, for mixtures, a compromise will be necessary. The analysis of anabolic steroids of abuse is shown. A detection limit of less than 200 pg/mL for testosterone from water is seen. Calibration for testosterone from water indicates a linear range to at least 1000 ng/mL, or three to four orders of magnitude. Several other steroids were extracted from urine at 10–100 ng/mL levels, and calibration for these compounds also exhibited linear behavior. The SPME with on-fiber headspace derivatization is a promising technique for the analysis of polar or nonvolatile compounds. © 1998 John Wiley & Sons, Inc. J Micro Sep 10: 551–556, 1998