The effect of pancreatic exocrine insufficiency (PEI) on protein malabsorption is little documented, partly due to methodological barriers. We aimed to validate biomarkers of protein malabsorption using a 15N test meal in a minipig model of PEI. Six pancreatic duct-ligated minipigs were used as a model of PEI and four nonoperated animals as a control. All animals were equipped with an ileocecal reentrant cannula. Minipigs were given a test meal containing [15N]casein. The PEI animals repeated the test three times, in the absence of any pancreatic enzymes, or after pancreatic substitution at two levels [A or B: 7,500 or 75,000 (lipase) and 388 or 3881 (protease) FIP U]. Ileal chyme, urine, and blood were collected postprandially. Nitrogen and 15N were measured in digestive and metabolic pools. We obtained a gradient of ileal protein digestibility from 29 ± 11% in PEI to 89 ± 6% in the controls and a dose- dependent response of enzymes. Insulin and gastric inhibitory polypeptide secretions were decreased by PEI, an effect that was counteracted with the enzymes at level B. The total recovery of 15N in urinary urea and plasma proteins was 14 ± 5.1% in the control group and decreased to 5.5 ± 2.1% by PEI. It was dose dependently restored by the treatment. Both 15N recovery in plasma and urine were correlated to protein digestibility. We confirm that the 15N transfer in those pools is a sensitive marker of protein malabsorption. Nevertheless, an optimization of the test meal conditions would be necessary in the view of implementing a clinical test. NEW & NOTEWORTHY We designed an intervention study to create a gradient of ileal protein digestibility in minipigs with pancreatic exocrine insufficiency and to validate reliable metabolic markers using a 15N oral meal test. 15N recovery in plasma proteins and to a higher extent in urine was sensitive to protein malabsorption. This test is minimally invasive and could be used to reveal protein malabsorption in patients.