Provided are methods for the detection and diagnosis of Hypothyroidism. The methods are based on the discovery that altered levels of selected analytes in sample fluid, typically blood samples, of patients are supportive of a diagnosis of Hypothyroidism. At least twenty-four new biomarkers for hypothyroidism are thus disclosed (singly or in any combination), Thyroid Stimulating Hormone, Interleukin-12p40, Tumor Necrosis Factor Alpha, Tissue Factor, Interleukin-15, Insulin, Immunoglobulin E, Growth Stimulating Hormone, Calcitonin, Prostate-Specific Antigen, Interleukin-4, Granulocyte Macrophage Colony Stimulating Factor, Matrix Metalloproteinase 9, Lymphotactin, Fatty Acid Binding Protein, Alpha Fetoprotein, Alpha-2 Macroglobulin, Serum Glutamic Oxaloacetic Transaminase, Matrix Metalloproteinase 3, Cancer Antigen 125, Mumps Antibody, Double Stranded DNA Antibody, Proliferating Cell Nuclear Antigen Antibody, Smith Antibody, or Herpes Simplex Virus 1 Glycoprotein D Antibody. Altogether the concentrations of one or more of these analytes, as well as Thyroid Stimulating Hormone, or any combination thereof, provide a sensitive and selective picture of the patient's condition, namely, whether the patient is suffering from Hypothyroidism. Kits containing reagents to assist in the analysis of fluid samples are also described.