Hypothyroidism is a disease caused by a dysfunction in the thyroid gland. The disease is divided into overt hypothyroidism (OHT) and subclinical hypothyroidism (SHT) according to the clinical signs and thyroid function hormones levels. Oxidative stress is a serious health situation related to many pathological conditions, including hypothyroidism. Oxidative damage can be caused at any cellular level including lipids, proteins, and nucleic acids. Protein carbonyl (PCO) is an indicator of protein damage caused by oxidative stress. Vitamin D perform a significant part in the human health, and its deficiency has become a global issue. We have tested the role of PCO and vitamin D in OHT and SHT types of hypothyroidism and investigated the possibility of using these two parameters in the screening of hypothyroidism. For this purpose 120 individuals, and included in three groups equally, OHT patients, SHT patients, and healthy control. The results were showed a significant (p<0.05) reduction in serum level of vitamin D for OHT patients compared to control and SHT groups, while the differences of vitamin D level were non-significant (p>0.05) between SHT and control groups. The level of PCO was elevated significantly in OHT and SHT groups compared to control, while SHT and OHT groups were showed a non-significant differences in PCO levels. Additionally, no correlation was found between vitamin D and PCO neither in OHT patients, nor in SHT patients. The sensitivity of vitamin D in the screening of OHT was good, but it was poor in SHT, while the sensitivity of PCO was good in both OHT and SHT groups. The results suggest a role of vitamin D deficiency in the pathophysiology of OHT, as well as elevated oxidative stress caused by protein carbonylation in both OHT and SHT groups.