Exocrine pancreatic insufficiency (EPI) is a condition characterized by a decreased synthesis and secretion of pancreatic enzymes, which results in weight loss, poor hair coat, and diarrhea. The diagnostic test of choice for EPI in domestic cats is feline serum trypsin-like immunoreactivity (fTLI). This paper details four tigers (Panthera tigris) with clinical signs compatible with EPI. On the basis of domestic cat reference ranges, fTLI assays for all four clinically affected tigers were diagnostic for EPI (median 1.0 µg/L; range 0.5–1.2 µg/L). All four tigers had a rapid clinical response to pancreatic enzyme supplementation. Serum from 10 clinically healthy tigers was submitted for the fTLI assay, for comparative purposes. The healthy tigers' fTLI assays were also within range for a diagnosis of EPI in domestic cats (median 3.1 µg/L; range 1.9–4.5 µg/L); however, clinically affected tigers had significantly lower serum fTLI concentrations than healthy tigers (P = 0.0058). Serum cobalamin was below the detection limit in both the affected and healthy tigers (<150 ng/L). Measuring fTLI appears to be a useful tool in the diagnosis of EPI-like syndrome in tigers. As in other species, EPI-like syndrome in tigers may also be associated with cobalamin deficiency.