An appropriate diagnostic evaluation is essential for the most appropriate treatment to be performed. Currently, macroprolactinemia is the third most frequent cause of nonphysiological hyperprolactinemia after drugs and prolactinomas. Up to 40% of macroprolactinemic patients may present with hypogonadism symptoms, infertility, and/or galactorrhea. Thus, the screening for macroprolactin is indicated not only for asymptomatic subjects but also for those without an obvious cause for their prolactin (PRL) elevation. Before submitting patients to macroprolactin screening and pituitary magnetic resonance imaging, one should rule out pregnancy, drug-induced hyperprolactinemia, primary hypothyroidism, and renal failure. The magnitude of PRL elevation can be useful in determining the etiology of hyperprolactinemia. PRL values >250 ng/mL are highly suggestive of prolactinomas and virtually exclude nonfunctioning pituitary adenomas (NFPAs) and other sellar masses as the etiology of hyperprolactinemia. However, they can also be found in subjects with macroprolactinemia, drug-induced hyperprolactinemia or chronic renal failure. By contrast, most patients with NFPAs, drug-induced hyperprolactinemia, macroprolactinemia, or systemic diseases present with PRL levels <100 ng/mL. However, exceptions to these rules are not rare. Indeed, up to 25% of patients harboring a microprolactinoma or a cystic macroprolactinoma may also have PRL <100 ng/mL. Falsely low PRL levels may result from the so-called “hook effect,” which should be considered in all cases of large (≥3 cm) pituitary adenomas associated with normal or mildly elevated PRL levels (≤250 ng/mL). The hook effect may be unmasked by repeating PRL measurement after a 1:100 serum sample dilution.