Clinical endocrinology has always had a close relationship with laboratory medicine. In fact, the quantification of hormones is of great importance for diagnosis, treatment, recurrence and patient's prognosis of endocrine disorders. This review dealt with the role of the laboratory in diagnosing endocrine pathologies related to adrenal gland, pituitary, gonads and thyroid. The measurements of the main hormones (17-hydroxyprogesterone, androstenedione, dehydroepiandrosterone sulphate, testosterone, estradiol, cortisol, aldosterone, metanephrines, thyroglobulin and insulin-like-growth factor-1) were described considering analytical characteristics but also some aspects of preanalytical and postanalytical steps. Traditionally, hormonal quantification is performed with immunoassays (IMAs), which have several advantages (i.e. limited training of technicians, high throughput, widely spread worldwide), but also some limitations on the accuracy of the results due mainly to cross-reactivity of IMA antibodies (i.e. steroid measurements) and protein interferences (i.e. heterophilic antibodies, antithyroglobulin antibodies in Tg measurements, etc.). In order to meet the need for clinicians to obtain ever more accurate results from laboratories, in recent decades, mass spectrometry (MS) has been developed. MS, in particular Liquid Chromatography-Tandem MS (LC-MS/MS), is a more complex but also more flexible technique able to guarantee greater specificity. It also provides multi-parameter quantification in the same analytical session delineating steroid profiles and, in some cases, defining the appropriate reference range for each hormone. Similar to IMAs, LC-MS/MS shows some inter-method variability, limiting the goal of standardization. However, several studies have recently demonstrated the possibility of reaching harmonization of this technology with good expectations for the future.