OBJECTIVE：To establish the content determination method of betaine in Lycium barbarum ，and to optimize the extraction method. METHODS ：HPLC method was used to determine the content of betaine in L. barbarum . The determination was performed on Waters Spherisorb NH 2 column with mobile phase consisted of acetonitrile- 0.01 mol/L monopotassiun phosphate aqueous solution （75∶25，V/V）at the flow rate was 0.7 mL/min. The detection wavelength was 195 nm，and column temperature was 30 ℃. The sample size was 10 μL. Using the content of betaine as index，on the basis of single factor tests ，L（9 34）orthogonal test design were used to select the methanol volume fraction ，extraction time and solid-liquid ratio of betaine in L. barbarum by ultrasonic extraction. The contents of betaine in 10 batches L. barbarum were determined ，and compared with the results of TLC included in 2015 edition of Chinese Pharmacopeia . RESULTS ：The linear range of betaine was 2.035-2 035.04 μg/mL（R2＝0.999 3）. The limits of detection and quantification were 0.410 μ g/mL and 2.051 μ g/mL，respectively. The average recovery were 97.41％-98.86％（RSDs were 0.8％-1.4％，n＝3）. RSDs of precision ，reproducibility and stability （24 h）tests were not higher than 1.2％. The optimal extraction method included solid-liquid ration of 1∶30（g/mL），ultrasonic extraction with methanol for 45 min. The average content of betaine in the extract from the three validation tests was 2.30％（RSD＝0.43％，n＝3）. The contents of betaine in 10 batches L. barbarum were 1.91％ -2.55％ ，which was no significantly different from the results of TLC （1.88％-2.60％）（RE were －1.92％-2.79％）. CONCLUSIONS ：The content determination method of betaine in L. barbarum was established successfully ，and the extract ion process was optinized.