Background: Macroprolactinemia is defined as hyperprolactinemia due to high levels of macroprolactin. The term 'macroprolactinemia' is used when the levels of macroprolactin exceed 60% of the total serum prolactin concentration estimated by polyethylene glycol (PEG) precipitation. Limited data is available on the burden of macroprolactinemia in Indian females. Aim: Find the prevalence of macroprolactin levels in females with hyperprolactinemia. Methods: The study was a hospital-based cross-sectional study with 98 subjects conducted in Central Clinical Lab of Adesh Institute of Medical Sciences and Research. Bathinda, a tertiary care teaching hospital in Punjab, India from July 2024 to April 2025. Subjects included females aged 18 years to 40 years attending the gynaecology OPD for infertility check up whose PrL levels were above the upper limits of laboratory reference range (25 ng/ml in females). Results: A total of 98 patients having a mean age of 29.2 ± 9.6 years were detected to have serum prolactin > 25 ng/mL over a period of July 2024 to April 2025. Further, 12 (12.2%) were detected to have macroprolactinemia. A total of 12 out of 98 patients detected to have macroprolactinemia had serum prolactin in the normal range post-PEG precipitation. Hence, 86 patients (87.75%) continued to have raised serum prolactin levels, even after post-PEG precipitation. Twelve patients were found to have macroprolactinaemia, and the prevalence of macroprolactinemia in hyperprolactinemia females came out to be 12.2%. Conclusion: The occurrence of macroprolactinemia in hyperprolactinemic females is not a rare phenomenon, with a prevalence of 12.2%. Differentiation of subjects with hyperprolactinemia secondary to macroprolactinemia from those with true hyperprolactinemia is clinically essential because the former condition is considered benign and generally requires no further investigation or medication. Further investigations like X-ray and MRI were dropped, which saved time and money of the patient.