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 a¹⁵N 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 and¹⁵N 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 of¹⁵N 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. Both¹⁵N recovery in plasma and urine were correlated to protein digestibility. We confirm that the¹⁵N 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 a¹⁵N 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.