Hypothyroidism is one of the most prevalent thyroid pathologies, which causes oxidative stress by disrupting antioxidant mechanisms. In mammals, the thyroid glands regulate metabolism, development, and growth. Dysfunction of the thyroid gland can result in hypothyroidism, hyperthyroidism, thyroiditis, and thyroid cancer. Whey protein is a widely consumed protein supplement containing abundant sulphur-containing amino acids and bioactive peptides. Here, we analysed the effect of whey protein on oxidative stress in hypothyroidism. In vivo studies were conducted in two phases for 30 and 90 days, respectively. Hypothyroidism was induced in Wistar albino rats by administering 0.05% propylthiouracil (PTU) through drinking water. Five hypothyroid groups and the normal control group were maintained in the first 30 day phase of the study. Among these, one group served as the induced control group, three groups received whey protein at different concentrations (100, 300, and 500 mg per kg body weight), and the last group received L-thyroxine (2 μg per 100 g body weight) as a positive medication. The activities of antioxidant enzymes, such as superoxide dismutase, catalase, glutathione peroxidase, and glutathione-S-transferase, were analysed, and the levels of total antioxidants, glutathione, and malondialdehyde were determined. Ca2+ ATPase and Na+/K+ ATPase activities were detected by estimating the inorganic phosphate content. Histopathological analysis was carried out on the thyroid and liver tissues of all groups. Antioxidant activity was notably increased for higher doses of whey protein compared to that in the diseased control group (p < 0.05). From this initial study, the dose that achieved the desired therapeutic effect was 500 mg kg-1, which was considered for the next 90 day phase of the study. The 90 day phase of the study was conducted with five groups: normal, whey protein-supplemented, hypothyroid, whey protein-supplemented hypothyroid, and levothyroxine-supplemented hypothyroid. All the PTU-treated groups showed degenerative alterations in thyroid histology. Whey protein supplementation causes a considerable decrease in MDA levels with an increase in the major antioxidant enzyme and ATPase activities, with p < 0.05. As a nutritional supplement, whey protein, at a 500 mg kg-1 dose, effectively boosts antioxidant activity without causing any toxicological concerns in long-term use.