Circadian rhythm (CR) in humans regulates the physical and mental changes that take place following rhythmic shifts of light and darkness. Dysregulation of CR is related to sleep disorders and other health problems including immune suppression, cancer, neurological and cardiovascular diseases. The two main hormones that maintain a balance to control the sleep-wake cycles are melatonin (MEL) and cortisol (COR). Here we describe a novel environmentally friendly low-density solvent-dispersive liquid-liquid microextraction-ultra-performance liquid chromatography-tandem mass spectrometry (LDS-DLLME-UPLC-MS/MS) method for simultaneous quantification of MEL, COR and their metabolites: 2-hydroxymelatonin (2-HMEL), 6-hydroxymelatonin (6-HMEL), cyclic-3-hydroxymelatonin (3-cHMEL), N-γ-acetyl-5-methoxykynurenamine (AMK), N-acetyl-N-formyl-5-methoxykynuramine (AFMK), N-acetylserotonin (NAS), 6-sulfatoxy melatonin (SaMT), 6-hydroxycortisol (6-HCOR), cortisol sulfate (CORS), cortisone (CON), β-cortol (CO) and β-cortolone (COL) in urine. Chemometrics approaches were applied for method optimization. The method was validated with the recovery of target analytes at 100 %, precision <16 % RSD and the absence of matrix effects. The method limits of quantification (LOQs) were from 0.013 ng mL for MEL to 0.79 ng mL for COL. This novel method was applied for the analysis of real urine samples. All targeted biomarkers were detected in urine samples, except AMK. MEL, 2-HMEL, 3-cHMEL, 6-HMEL, AFMK, NAS, COL and CO were found predominantly as glucuronidated conjugates, whereas SaMT, CORS and 6-HCOR were detected in free form in urine. We observed remarkable variation in profiles of MEL and COR metabolites in first morning void, daytime and nighttime urine samples, suggesting that this method can be applied for monitoring circadian rhythm in adults and children. The most sensitive urinary biomarkers of CR are 2-HMEL, 6-HMEL, SaMT, COR, 6-HCOR and CON.