The consumption of psychostimulant amphetamine-like drugs has increased significantly in recent years. Some MDMA metabolites are probably involved in the neurotoxicity and neurodegeneration caused by prolonged use rather than MDMA itself. We recently developed a method to analyze MDMA and its five main metabolites in rat plasma [7]. We have now fully validated this method to the quantification of these drugs in rat urine. We extracted MDMA and its metabolites with Oasis WCX cartridges, separated them on a Nucleodur C18 analytical column and quantified them by ion-trap mass spectrometry. Linearity was excellent: 12.5–1250ng/mL urine for HMA, HMMA, MDA and MDMA, 25–2500ng/mL for HHMA, and 150–7500ng/mL for HHA (r 2 >0.993 for all analytes). The lower limits of quantification were 12.5ng/mL urine for MDMA, MDA, HMA and HMMA, 25ng/mL for HHMA and 150ng/mL for HHA. Reproducibility was good (intra-assay precision=1.7–6.1%; inter-assay precision=0.6–5.7%), as was accuracy (intra-assay deviation=0.1–4.8%; inter-assay deviation=0.7–7.9%). Average recoveries were around 85.0%, except for HHMA (66.2%) and HHA (53.0%) (CV<8.3%). We also checked the stability of stock solutions and the internal standards after freeze-thawing and in the autosampler. Lastly, we measured the MDMA, MDA, HHMA, HHA, HMMA and HMA in urine samples taken over 24h from rats given subcutaneous MDMA.