Testosterone, the most abundant androgen in men, is a steroid hormone that is synthesized predominantly by the testes. In women, minor amounts are synthesized in the ovaries. Androgen precursors are also produced and secreted from the adrenal glands in both sexes, where they undergo peripheral conversion to testosterone. Circulating concentrations are approximately 15-25 times higher in adult men compared to women. Maintenance of these levels is necessary for development and maintenance of secondary sexual characteristics, libido, growth, prevention of osteoporosis, and most importantly in men, spermatogenesis. Most testosterone circulates tightly bound to sex hormone-binding globulin (SHBG) or weakly bound to albumin. A minor amount circulates as free testosterone, and it is believed that this is the metabolically active fraction. Measurement of free testosterone is important in the diagnosis of many diseases, most importantly disorders of androgen deficiency in men (i.e., hypogonadism) and androgen excess in women (i.e., polycystic ovary syndrome and hirsutism). Many methodologies are available for free testosterone measurement including the reference methods (equilibrium dialysis and ultrafiltration), analog immunoassay, and calculated free testosterone based on measurement of total testosterone, SHBG, and albumin. Moreover, measurement of bioavailable testosterone, a combination of albumin-bound and free testosterone, also has clinical utility and can be measured by selective protein precipitation or calculation. In this review, the advantages and limitations of each of these methods will be discussed in the context of clinical utility and implementation into a routine hospital laboratory. Furthermore, up and coming methodologies for free testosterone measurement, including liquid chromatography-tandem mass spectrometry, will also be discussed.