Biomarkers of urinary melatonin represent an attractive non-invasive method for enhancing breast cancer diagnosis and follow-up, as they reflect combined circadian physiology in a readily accessible and repeatable urine test. There has been a growing interest in this field, driven by the understanding that melatonin plays a role in regulating the endocrine system, influencing sleep patterns, managing circadian rhythms, affecting exposure to night-time light, and impacting the body's response to cancer and its treatment. The urinary analyte 6-sulfatoxymelatonin has become the most viable surrogate of systemic nocturnal melatonin secretion, due to its increased stability, higher concentration, and suitability for timed, overnight, first-morning, and 24-h urine collection paradigms. Epidemiological, case-control, and longitudinal studies indicate that changes in urinary melatonin levels may be linked to breast cancer risk, disease burden, treatment-associated circadian disruption, and survival-associated sleep impairment. This review aims to present a structured review of the biological foundation, measurement, pre-analytical and analytical determinants, and clinical evidence of urinary melatonin analysis in breast cancer. We compared parent melatonin and 6-sulfatoxymelatonin, assessed immunoassays and chromatographic systems, and discussed significant confounders, such as age, menopausal status, renal function, chronotype, light-at-night exposure, sleep behavior, diet, medications, and sample handling. We further refer to identify the weaknesses that at present clinical translation, such as pre-analytical variability, heterogeneity of assays, absence of reference intervals, and inadequate prospective validation. We believe that the most likely future use of urinary melatonin is as a biomarker component in circadian-informed strategies, where it supplements tumor-specific biomarkers by indicating host physiology, biological time, and treatment-associated circadian disruption.
