Polycystic ovary syndrome (PCOS) is a common and complex disorder characterized by endocrine, metabolic, and reproductive disturbances. Both the clinical and laboratory features of the syndrome are remarkably heterogeneous and may change even in a single patient over time. Laboratory investigations are used in PCOS mainly for the determination of biochemical hyperandrogenemia and ovulatory dysfunction. These tests are also helpful for the exclusion of other related androgen excess disorders. Measurement of serum-free testosterone levels by sensitive methods or calculation of free androgen index, in addition to measurement of dehydroepiandrosterone sulfate. are used for the determination of hyperandrogenism. Serum levels of luteinizing hormone or the luteinizing hormone/follicle stimulating hormone ratio are not recommended for the routine evaluation of PCOS, whereas luteal-phase progesterone measurements might be helpful to confirm ovulatory function in hyperandrogenic patients with apparently regular menses. Initial work-up of a patient with PCOS includes, at a minimum, thyroid-stimulating hormone, prolactin, and basal or stimulated 17(OH) progesterone levels to exclude thyroid dysfunction, hyperprolactinemia, and nonclassical congenital adrenal hyperplasia, respectively. If other rare disorders with similar clinical presentations are suspected, further hormonal and biochemical evaluation would be necessary.