Tramadol is a widely prescribed analgesic used in the treatment of moderate to severe pain and as an alternative to opiates.Analytical procedures for the determination of tramadol and its major metabolites, O-desmethyltramadol (M1) and N-desmethyltramadol (M2) in human urine have been developed and validated using gas chromatography–mass spectrometry (GC/MS). Sample preparation involved liquid–liquid extraction with tert.-butylmethyl ether (MTBE) and back extraction with 0.1N hydrochloric acid. Proadifen (SKF525A) was selected as internal standard (IS). Extraction efficiencies of tramadol, M1 and M2 were 101.88%, 98.51% and 108.65% respectively. For qualitative analysis we operated in scan mode and for quantitative analysis in SIM mode, selecting the ions m/z 58 for tramadol and M1, m/z 188 for M2 and m/z 86 for IS. The calibration curves were linear (r2>0.997) in the concentration range 10–1000 ng/mL for all compounds. The lower limit of quantitation was 10 ng/mL for tramadol and M1, and 20 ng/mL for M2. The intra-day precision of the assay (n=6) for the measurement of QC samples at three concentrations was in the range 2.29–5.82%, 1.29–6.88% and 1.46–6.78% for tramadol, M1 and M2, respectively; inter-day precision was in the range 2.12-3.73%, 1.14-6.50% and 3.04-5.48% for tramadol, M1 and M2, respectively; the intra-day accuracy was in the range 90.40–100.87%, 94.81–107.06% and 99.94–103.20% for tramadol, M1 and M2, respectively. Data on solution stability at room temperature and 4°C in urine samples, freeze–thaw-stability, sample extract stability as well as dilution factor and matrix effects have been evaluated andwill be presented. The application of the assay was demonstrated by simultaneous measurement of urine concentrations of T, M1, and M2 in samples from healthy volunteers after the administration of a 50 mg oral doses of tramadol.