OBJECTIVE To analyze the measurement uncertainty in determination of levodopa Me ester hydrochloride in plasma by LC-MS/MS.METHODS Thermo BDS HYPERSIL C18 column (4.6 mm × 100 mm, 2.4 μm) was used, the mobile phase consisted of methanol and ammonium acetate (10 mmol·L-1, pH = 5), using gradient elution, and the flow rate was 0.4 mL·min-1.Mass spectrometry was performed in multiple reaction monitoring (MRM) mode, with m/z 212.4→152.3 for levodopa Me ester hydrochloride and m/z of 166.4→148.3 for internal standardA math. model was established to analyze the source of uncertainty.The various factors influencing the uncertainty in the whole process of determination, including precision, weighing, solution preparation, plasma containing drug preparation, plasma extraction, the apparatus and calibration curves fitting, were all analyzed and estimatedThe uncertainty and synthetic uncertainty of each variable were calculatedThe expanded uncertainty was analyzed with all the components.RESULTS When the confidence probability P = 95%, the expanded uncertainty for low, medium and high (0.4, 4, 32 ng·mL-1) concentration of zidovudine were 0.10, 0.23, 1.62 ng·mL-1, resp.CONCLUSION This method is suitable for the uncertainty evaluation for the determination of levodopa Me ester hydrochloride concentration in human plasma by LC-MS/MS, and provides reference for uncertainty evaluation of complex biol. matrix anal.