The near infrared (NIR) spectra of Liuyi San samples were collected during the mixing process and the quantitative models by PLS (partial least squares) method were generated for the quantification of the concentration of glycyrrhizin. The PLS quantitative model had good calibration and prediction performances (r(cal) 0.998 5, RMSEC = 0.044 mg · g(-1); r(val) = 0.947 4, RMSEP = 0.124 mg · g(-1)), indicating that NIR spectroscopy can be used as a rapid determination method of the concentration of glycyrrhizin in Liuyi San powder. After the validation tests were designed, the Liao-Lin-Iyer approach based on Monte Carlo simulation was used to estimate β-content-γ-confidence tolerance intervals. Then the uncertainty was calculated, and the uncer- tainty profile was drawn. The NIR analytical method was considered valid when the concentration of glycyrrhizin is above 1.56 mg · g(-1) since the uncertainty fell within the acceptable limits (λ = ± 20%). The results showed that uncertainty assessment can be used in NIR quantitative models of glycyrrhizin for different concentrations and provided references for other traditional Chinese medicine to finish the uncertainty assessment using NIR quantitative analysis.