Background Certain amino acids are decreased in humans with inflammatory bowel disease (IBD) and supplementation with the same amino acids has shown beneficial effects in animal models of IBD. Currently, the amino acid status of dogs with protein‐losing enteropathy (PLE) is unknown. Hypothesis/Objective To determine if serum amino acid concentrations are abnormal in dogs with PLE and correlated with clinical and laboratory variables and outcome. Animals Thirty client‐owned dogs diagnosed with PLE and 12 apparently healthy dogs seen at Bristol Veterinary School. Methods Retrospective study using stored residual serum from fasted dogs with PLE, collected at the time of diagnostic investigation and from apparently healthy dogs. Serum was analyzed for 30 amino acids using an automated high‐performance liquid chromatography amino acid analyzer. Results Serum tryptophan concentrations were significantly decreased in dogs with PLE (median, 22 nmol/mL; range, 1–80 nmol/mL) compared with apparently healthy control dogs (median, 77.5 nmol/mL; range, 42–135 nmol/mL, P < .001). There were no significant differences in the remaining 29 serum amino acids between dogs with PLE and apparently healthy. Serum tryptophan concentrations were also significantly correlated with serum albumin concentrations in dogs with PLE (P = .001, R2 = 0.506). Conclusions and Clinical Importance Decreased serum tryptophan concentration might play a role in the pathogenesis of canine PLE or be a consequence of the disease.