Pharmacokinetic studies require sensitive analytical methods to allow the determination of low concentrations of drugs and metabolites. When drugs present an asymmetric center, the enantiomeric determination of the compounds of interest should be performed. The method developed is based on on-line LC-MS-MS using atmospheric pressure chemical ionization as an interface determination of enantiomers of tramadol (T) and its active metabolite O-desmethyltramadol (ODT) in human plasma. This determination is preceded by an off-line solid-phase extraction (SPE) on disposable extraction cartridges (DECs), performed automatically by means of a sample processor equipped with a robotic arm (ASPEC system). The DEC filled with ethyl silica (50 mg) was first conditioned with methanol and water. The washing step was performed with water and the analytes were finally eluted by dispensing methanol. The collected eluate was then evaporated to dryness before being dissolved in the LC mobile phase and injected into the LC system. The enantiomeric separation of tramadol and ODT was achieved on a Chiralpak AD column containing amylose tris-(3,5-dimethylphenylcarbamate) as chiral selector. The mobile phase was isohexane-ethanol-diethylamine (97:3:0.1, v/v). The LC system was then coupled to a tandem mass spectrometry system with an APCI interface in the positive ion mode. The chromatographed analytes were detected in the selected reaction monitoring mode. The MS-MS ion transitions monitored were 264-->58 for tramadol, 250-->58 for ODT, and 278-->58 for ethyltramadol, used as internal standard. The method was validated. The recoveries were around 90% for both T and ODT. The method was found to be linear for each enantiomer of both compounds (r2>0.999). The mean RSD values for repeatability and intermediate precision were 3.5 and 6.4% for T enantiomers and 5.0 and 5.6% for ODT enantiomers, respectively. Moreover, the method was found to be selective towards other metabolites, N-desmethyltramadol and N,O-desmethyltramadol (NODT). The method developed was successfully used to investigate plasma concentration of enantiomers of T and ODT in a pharmacokinetic study.