Hypothyroidism is a clinical condition caused by the deficiency of thyroid hormones that are T4 and T3 due to an increase in serum TSH level. The upper and lower limit of T4 and TSH helps to identify the disease. The metabolic pathways are important to know for diagnostic tests. By conducting different biochemical tests, a proper diagnosis can be performed when hypothyroidism is suspected clinically. Previously, many assays were performed just to detect the disease but recent tests are in both direct and indirect categories. Direct tests are purposely enough to detect the deficiency of thyroid hormones just like T3, T4, serum TSH, free-T4, free-T3, T4 resin uptake, free T4 index, T4 binding globulin, and anti-TPO. All these tests are performed considering the reference ranges of hormones and the discovery that lower and higher TSH readings, even within the standard range, could predict future hypothyroidism, respectively, while the connection is stronger for hypothyroidism, supports the significance of diagnosing moderate thyroid status problems. If needed, one can also consider the radiological test that is a radioactive iodine uptake test. Other biochemical tests are considered as indirect because these tests actually confirm other changes in the body due to hypothyroidism such as lipid profile tests (TC, TAG, HDL-C, TC/HDL-C, and TAG/HDL-C), cytokine tests (interleukin-6, TNF-α, visfatin, and leptin) and other regular tests like iron deficiency test, hemoglobin test, ferritin, and TIBC. This article carries brief information regarding all the tests mentioned above and their purpose of conduction in hypothyroidism disease.