A rapid, sensitive and accurate liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for the simultaneous determination of tramadol and its active metabolite, O-desmethyltramadol in human plasma is developed using propranolol as internal standard (IS). The analytes and IS were extracted from 200 microL aliquots of human plasma via protein precipitation using acetonitrile. Chromatographic separation was achieved in a run time of 2.0 min on an Aquasil C18 (100mm x 2.1mm, 5microm) column under isocratic conditions. Detection of analytes and IS was done by tandem mass spectrometry, operating in positive ion and multiple reaction monitoring (MRM) acquisition mode. The method was fully validated for its selectivity, sensitivity, linearity, precision and accuracy, recovery, matrix effect, ion suppression/enhancement, stability and dilution integrity. A linear dynamic range was established from 1.0 to 600.0ng/mL for tramadol and 0.5-300.0ng/mL for O-desmethyltramadol. The method was successfully applied to a bioequivalence study of 200mg tramadol tablet formulation in 27 healthy Indian male subjects under fasting condition.