This study aimed to clarify the laws of glutamine tablets absorption, distribution, and metabolism in Beagles and to provide a basis for formulating dosing regimens. Twelve healthy Beagles were enrolled the absolute bioavailability study with a crossover design. Glutamine tablets (240 mg/kg b.w.) or glutamine sterile solution (60 mg/kg b.w.) were administered. A method for the determination of glutamine in Beagles' plasma by UPLC‐MS/MS was established, with high sensitivity, specificity, and simplicity. Based on the study of endogenous glutamine concentration, the mean concentration of the four time points before drug administration was selected as the background concentration of glutamine. Pharmacokinetic parameters were calculated by non‐compartment model. The Cmax of glutamine was 136.11 ± 72.51 μg/ml, Tmax was 0.85 ± 0.29 h, and t1/2λz was 0.42 ± 0.27 h after oral administration. The AUC0−t of glutamine was 116.30 ± 75.15 h·μg/ml vs. 44.55 ± 22.48 h·μg/ml following oral and IV administration, respectively, with an absolute bioavailability of 64.74% ± 19.18%. The results showed glutamine was quickly absorbed and eliminated in Beagles with high bioavailability. Therefore, glutamine is suitable to be prepared as oral tablets and recommended to shorten the dosing interval. [ABSTRACT FROM AUTHOR]