Steroid hormones are synthesised from cholesterol. The majority of these powerful molecules are produced by the adrenal glands, testes or ovaries. They fulfil a wide range of physiological functions after binding to specific cell receptors of various target organs. Mainly they are involved in the carbohydrate, lipid and protein metabolism, stress response and in the regulation of the water and mineral balances, in reproductive processes and the development of secondary sex characteristics. Consequently steroid hormones are also involved in a variety of diseases, like adrenal tumors, the Adrenogenital syndrome (CAH), the Cushing’s syndrome or the Polycystic Ovarian Syndrome. The quantitative profiling of steroids is essential in diagnosis and evaluation of pathologic conditions and therapeutic responses.