Abstract: The study aims to solve the instability problem of methylphenidate (MPH) in plasma, and establish a LC-MS/MS method for simultaneous determining of MPH in human plasma. The stabilities of MPH in different media were studied, and the degradation characteristics of MPH in these media were also investigated by HPLC and LC-MS/MS. To a 200 µL aliquot of freshly collected plasma sample, 10 µL 2% formic acid was added immediately to prevent the hydrolysis of MPH in human plasma samples. Chromatographic separation was performed on a Sapphire C₁₈ column using the mobile phase of methanol-5 mmol·L^-1 ammonium acetate buffer solution containing 0.1% formic acid (46:54). MPH was quantified by tandem mass spectrometry operating in positive electrospray ionization mode with multiple reaction monitoring. The detection used the transitions of protonated molecules at m/z 234.2 → 84.1 for MPH and m/z 260.3 → 183.1 for propranolol (IS), separately. The intra-and inter-assay precisions were all below 5.0%. The accuracies were all in standard ranges. The linear calibration curve was obtained in the concentration range of 0.035-40 ng·mL^-1. The methods fulfilled the demand. The method was used to determine the concentration of MPH in human plasma after a single dose of 36 mg MPH tablet to 6 healthy Chinese volunteers. The method is suitable for the precisely determination of MPH and for pharmacokinetic study of MPH in human plasma.