For many years it was believed that only amino acids, dipeptides and tripeptides could be absorbed and reach the bloodstream. Nowadays, the bioavailability of oligopeptides is also being considered, requiring further research. Hence, in this pilot study, the activity of the brush border enzymes on undigested (WPH) and digested (ID) whey hydrolysates and the absorption of the resultant peptides through the proximal jejunum of a 7-week old piglet in Ussing chamber model was investigated. Among all samples, 884 oligopeptides were identified. The brush border peptidase activity was intense in the first 10 min of the experiment generating several new peptides in the apical compartment. Within ID, 286 peptides were detected in the basolateral compartment after 120 min of experiment originating from β-lactoglobulin (60%) and β-casein (20%). Nevertheless, only 0.6 to 3.35% of a specific peptide could pass through the epithelial barrier and reach the basolateral compartment. The present study showed for the first time the ex-vivo transepithelial jejunum absorption of whey oligopeptides. It also confirmed the proteolytic activity of brush border enzymes on these oligopeptides, representing an important step towards the bioavailability of these peptides with biofunctional potential. Data from this dataset were acquired on a Qexactive mass spectrometer coupled to a NanoRSLC liquid chromatography with a Proxeon Electrospray interface.