Background: The thyroid function of the dog has been little studied in comparison with that of the human. This may have resulted, in part, from the apparent lack of reporting of thyroid-related disorders in the Indian canine population. There is, therefore, a need for a more detailed study of thyroid hormone levels (establish reference interval) in normal dogs using a specific and accurate technique like radioimmunoassay (RIA). Methods: Total 192 healthy dogs from the clinical setup of constituent colleges of MAFSU (viz. Mumbai, Nagpur, Parbhani, Shirwal, Udgir and Akola) were ethically enrolled to examine thyroid hormones levels (TT3, TT4 and FT4). Thyroid hormones were estimated using RIA. Result: The results of the present study recorded mean serum TT3, TT4 and FT4 values in healthy dogs as 1.29±0.04 nmol/l, 28.17±1.18 nmol/l and 13.03±0.68 pmol/l respectively. The reference interval (25th to 75th percentile) for TT3, TT4 and FT4 was found to be 0.88-1.51 nmol/l, 15.70-35.29 nmol/l and 7.80-14.75 pmol/l respectively. The median for TT3, TT4 and FT4 was found to be 1.13 nmol/l, 24.54 nmol/l and 10.00 pmol/l respectively. The baseline serum thyroid hormone concentration would be valuable for identifying thyroid dysfunction in dogs.