Abstract: To evaluate the uncertainty of determination of Salidroside in proficiency testing samples of traditional Chinese medicine by high-performance liquid chromatography (HPLC), the content of salidroside was determined by the content determination method of Rhodiola in the first part of Pharmacopoeia of the People's Republic of China (2015 Edition). According to JJF 1059.1-2012 "Evaluation and Expression of Uncertainty in Measurement", the sources of uncertainty in the determination of Salidroside in two samples of Rhodiola powder No. 153 and No. 275 were analyzed. The mathematical model is established to quantify the uncertainty components, and the combined uncertainty and expanded uncertainty are calculated and compared with the results of capability verification. The result of No.153 Rhodiola powder sample was (1.41±0.05) % with k=2, the result of No.275 Rhodiola powder sample was (2.58±0.08)% with k=2, and the result of the proficiency test was satisfactory. The uncertainty of salidroside content in the two samples mainly came from repeatability tests, standard curve fitting, and dilution of reference series solution. This study can provide a reference for evaluating the quality of salidroside content determination results and controlling the internal quality of the laboratory. At the same time, uncertainty evaluation and proficiency testing results showed that the laboratory was capable of conducting the determination of salidroside in Chinese medicinal materials at the concentration level from 102.8 μg/mL to 1028 μg/mL and ensure the reliability of data.