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Objective: The aim of this study was to investigate the urine prolactin and serum/urine prolactin ratio for accuracy diagnosis of macroprolactinemia. Methods: In the retrospective cross-sectional analysis, prolactin levels (high or normal) in the reproductive period of men and women were included in the study. Polyethylene glycol (PEG) precipitation method was used for the detection of macroprolactinemia. Then, patients were divided into three groups as macroprolactinemia, prolactinoma and healthy control group. In patients, prolactin values in spot urine with simultaneous serum prolactin values were calculated. The non-parametric Kruskal-Wallis test was used to compare the groups. The receiver-operating characteristic (ROC) curve was determined to evaluate the predictive power of serum/urine prolactin ratio. Results: A total of 41 patients were included in the study. Female/male:36 (87.8%)/5 (12.2%). Urinary prolactin median(minimum-maximum) values were macroprolactinemia, prolactinoma and control group, respectively; 0.06(0.05-0.10), 0.11(0.02-0.95), 0.08(0.05-0.25) ng/ml. Serum/urine ratio median (minium-maximum) values were macroprolactinemia, prolactinoma, and control group, respectively; 633(51-1032), 990(104-9635), 395.5(138-953). When the groups were compared, the patients with prolactinoma had higher urinary prolactin levels(p <0.01). Serum/urine prolactin ratio was found to be the highest in prolactinoma patients and the lowest in the control group and a significant difference was observed in groups (p <0.01). ROC analysis(control-macroprolactin) for serum/urine prolactin ratio (Sensitivity 84.6 specificity 93.7 cut off >549,5 AUC=0.83) p<0.01 Conclusion: Urinary prolactin level and serum/urine prolactin ratio may be used in diagnosis of macroprolactinemia. [ABSTRACT FROM AUTHOR]