Objective: This study aimed to evaluate the uncertainty of determination by high performance liquid chromatography (HPLC) for the content of adevantame, a synthetic sweetener illegally added in syrup drugs. Methods: The sample is diluted with solvent, and the content of adevantame in the sample was determined by HPLC. Through the analysis of the factors affecting the uncertainty, such as the preparation of the reference solution, curve fitting, sample weighing, sample volumetric determination and instrument precision, the uncertainty was synthesized of each component, and the extended uncertainty is calculated. Results: The expanded uncertainty is 1.8 μg/mL. The influence factors from large to small was as follows: repeated sample determination, standard curve fitting, standard solution preparation, sample solution preparation. Conclusion: The established uncertainty evaluation method is accurate and reliable, and truly reflects the source and magnitude of uncertainty.