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[Display omitted] • Lycium barbarum L. berries as a source of valuable bioactive compounds. • Response Surface Methodology enabled the optimization of the extraction process. • Phenolic acids, flavanols, and phenylamides were detected. • The extract proved to be safe for intestinal application at doses up to 500 ug/mL. • Different extract's compounds permeated the cell monolayer. Lycium barbarum L. berries have a remarkable chemical composition and extensive biological activities, being a valuable component of health and nutraceutical practices. Nevertheless, a deep insight on the intestinal permeation of the pro-healthy bioactive compounds is urgently needed to predict the real effects on human body. This study attempted, for the first time, to optimize the Ultrasound-Assisted Extraction (UAE) of goji berries using a Response Surface Methodology approach and establish the intestinal permeation of the principal pro-healthy compounds. The optimal extraction conditions were a solid:liquid ratio of 8.75 % for 56.21 min, using an intensity of 59.05 W/m2. The optimal extract displayed a remarkable antioxidant capacity, with LC/DAD-ESI-MS analysis unveiled a diverse phytochemical profile, encompassing different compounds (e.g. glu-lycibarbarspermidine F, 2-glu-kukoamine, rutin, 3,5-dicaffeoylquinic acid). The intestinal co-culture model demonstrated that glu-lycibarbarspermidine F (isomer 2) (73.70 %), 3,5-dicaffeoylquinic acid (52.66 %), and isorhamnetin-3- O -rutinoside (49.31 %) traversed the intestinal cell layer, exerting beneficial health-promoting effects. [ABSTRACT FROM AUTHOR]