The purpose of this study was to validate a thyroid-stimulating hormone (TSH) assay in a model of equine hypothyroidism. Thyrotropin-releasing hormone (TRH) stimulation tests were performed in 12 healthy adult mares and geldings, aged 4 to greater than 20 years, before and during administration of the antithyroid drug propylthiouracil (PTU) for 6 weeks. Serum concentrations of equine TSH, total and free thyroxine (T4), and total and free triiodothyronine (T3) were measured. Before PTU administration, mean ± standard deviation baseline concentrations of TSH were 0.40 ± 0.29 ng/mL. TSH increased in response to TRH, reaching a peak concentration of 0.78 ± 0.28 ng/mL at 45 minutes. Total and free T4 increased from 12.9 ± 5.6 nmol/L and 12.2 ± 3.5 pmol/L to 36.8 ± 11.4 nmol/L and 23.1 ± 5.9 pmol/L, respectively, peaking at 4–6 hours. Total and free T3 increased from 0.99 ± 0.51 nmol/L and 2.07 ± 1.14 pmol/L to 2.23 ± 0.60 nmol/L and 5.78 ± 1.94 pmol/L, respectively, peaking at 2–4 hours. Weekly measurements of baseline TSH and thyroid hormones during PTU administration showed that total and free T3 concentrations fell abruptly and remained low throughout PTU administration. Total and free T4 concentrations did not decrease dramatically until weeks 5 and 4 of PTU administration, respectively. A steady increase in TSH concentration occurred throughout PTU administration, with TSH becoming markedly increased by weeks 5 and 6 (1.46 ± 0.94 ng/mL at 6 weeks). During weeks 5 and 6 of PTU administration, TSH response to TRH was exaggerated, and thyroid hormone response was blunted. Results of this study show that measurement of equine TSH in conjunction with thyroid hormone measurement differentiated normal and hypothyroid horses in this model of equine hypothyroidism.