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[Objective] To establish a mathematical model of uncertainty assessment for lysine content in Xianyu 335 corn by the amino acid assay, completely concerning the sources of uncertainty in the measurement process. [Methods] Analyze and measure the main source of uncertainty, and evaluate and calculate the each component of uncertainty. [Results] For 0.1055 g sample, the lysine determination in Xianyu 335 showed a good linear relationship. [Conclusion] This method is suitable for the Uncertainty Evaluation of lysine measurement in Xianyu 335 by amino acid assay.