Polyphenols are the important active compounds present in the fruits of Lycium barbarum L. The objectives of this study were to develop an ultra-high performance liquid chromatography with UV detection (UHPLC-UV) method for qualitative and quantitative analyses of phenolic acids and flavonoids in fruits of L. barbarum L. The isolation and enrichment of polyphenols from fruits were carried out by ultrasound-assisted extraction (UAE) in conjunction with solid-phase extraction (SPE). The analytes were separated on a Poroshell 120 EC-C18 column using a binary mobile phase composed of aqueous 0.05 % trifluoroacetic acid and acetonitrile in the gradient elution mode. Under these conditions, phenolic acids and flavonoids were separated in 11 min. The selectivity of the developed UHPLC-UV method was confirmed by comparison with ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) analysis. The validation parameters such as linearity, sensitivity, precision, and accuracy were found to be highly satisfactory. The optimized method was successfully applied to the analysis of polyphenols in fruits of L. barbarum L. Additionally, their free radicalscavenging activity was assessed by 1,1-diphenyl-2-picrylhydrazyl radical (DPPH • ).