Flupirtine (FLU) is a nonopioid analgesic drug with no antipyretic or antiphlogistic effects labeled for humans. It does not induce the side effects associated with the classical drugs used as pain relievers (Non steroidal antiinflammatory drugs and opioids) in human beings. The aim of this study was to evaluate the pharmacokinetic profiles of FLU after IV and PO administrations in healthy donkeys. Six Amiata breed adult jennies were randomly assigned to two treatment groups using an open, 2 × 2 Latin-square crossover study design. Group 1 (n = 3) received a single dose of 1 mg/kg of FLU injected IV into the jugular vein. Group 2 (n = 3) received FLU (5 mg/kg) via nasogastric tube. The washout period was 1 week. Blood samples (5 mL) were collected at 0.083, 0.25, 0.5, 0.75, 1, 1.5, 2, 4, 6, 8, 10, 24, 36, and 48 hours, and plasma was then analyzed by a validated high-performance liquid chromatography method. No adverse effects were noticed in either administration group. After IV and PO administrations, FLU was detectable in plasma for up to 24 hours. The mean elimination half-life was longer after PO (10.81 hours) than after IV (0.90 hours) administration. The clearance was fast, and the area under the plasma concentration–time curve was small, findings consistent with a low PO bioavailability of about 20%. The pharmacokinetic trend of FLU in donkeys was different from those earlier reported in cats and dogs. Further studies are needed to understand if this active ingredient may be used in donkeys.