Abstract Background: Immunoassays show variability in the detection of macroprolactin. The aim of this study was to detect the frequency of macroprolactinemia in hyperprolactinemic patients and the problems encountered in routine clinical practice. Methods: The screening of macroprolactinemia was performed by precipitation with polyethylene glycol (PEG) in 900 patient samples with hyperprolactinemia over a period of approximately 6 months. Recovery values of less than 40% and greater than 60% were considered as macroprolactinemia and predominantly monomeric prolactin (PRL), respectively. Results: A total of 900 (17.9%) of the 5007 PRL results were out of reference range. Thirty-one (3.4%) of the patients had less than 40% recovery after screening of all patients with hyperprolactinemia. However, the macroprolactin test was requested by clinics from only 171 patients and seven of these patients had less than 40% recovery. We also detected predominantly macroprolactin in 24 samples, overlooked in routine practice. The patients with PRL above 100 ng/mL had no macroprolactinemia. Conclusions: The screening for macroprolactinemia of hyperprolactinemic patients who have <100 ng/mL and also with unexplained hyperprolactinemia should be the first approach before any further research or treatment is initiated. Thus, unnecessary test repetition, investigation and inappropriate treatment can be avoided. Each laboratory should inform clinicians about the frequency of macroprolactinemia.