In Vietnam, Physalis angulata L. has been widely used as a traditional medicine to treat fever, anti-inflammatory, and expectorant. Currently, there have been studies on the content of chemical composition especially physalin with anti-diabetic, anti-inflammatory, antibacterial, prevent cancer. This study developed a reliable and sensitive method to determine and validate simultaneous Physalin B and Physalin D in Physalis angulata L.. The QuEChERS method was used for sample preparation from leaf matrices and quantified by using High-performance liquid chromatography coupled with a diode-array detector. The method of research was validated under AOAC and ICH guidance. Chromatography conditions include Agilent C18 column (250mm × 4,6mm; 5µm) with a gradient mode using acetonitrile – methanol-water as mobile phase. The recovery of the method ranged from 94.21 – 105.93% and RSD was from 1.20 – 2.31%, the LOD, and LOQ were 0.4 mg/kg – 2.4 mg/kg, respectively. The results of the study showed that the proposed the new
QuEChERS method for quantification of Physalin B and D in Physalis angulata L. in Vietnam.