Donkey's milk is well tolerated by human infants with cow's milk allergy and is useful in the treatment of human immune-related diseases and in the prevention of atherosclerosis. Thyroid hormones (TH) stimulate lactation and active triiodothyronine (T3) in colostrum and milk could take paracrine action supporting lactogenesis in the mother, and play physiological roles for the suckling offspring (systemic or within the gastrointestinal tract). The aims were to measure TH concentrations in donkey blood and milk, validate ELISA methods, evaluate the effects of sample collection and post-collection handling and the stability of TH in milk and blood serum and plasma samples. In milk and blood samples obtained from lactating jennies total concentrations of TH were assayed using competitive-type ELISA kits. Good validation results were obtained for both TH concentrations in blood serum and plasma and T3 in milk samples extracted with cold (-20°C) ethanol alkalinized (pH 9·0) with NH4OH. In most of the milk extract samples, thyroxine (T4) concentrations resulted below the sensitivity threshold. Intra- and inter-assay coefficients of variations of TH concentrations in different blood and milk samples were below 10%. Parallelism tests gave displacement lines parallel to those of the calibrators for both TH in blood serum and plasma and for T3 in milk extracts. Mean recovery rates were between 95% and 123%, but the concentration values approaching the highest calibrators were overestimated. Therefore, serum and plasma samples for T3 assay must be previously diluted with buffer. Both TH concentrations in blood serum and plasma and T3 in milk did not change during storage for up to 6 months at -20°C. In conclusion, the ELISA methods tested in the present study are suitable for determination of both TH concentrations in donkey blood samples, and for T3 measurement in milk, after extraction with cold alkaline ethanol.