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Melatonin (N-acetyl-3-(2-aminoethyl)-5-methoxyindole) is biologically active as a neurohormone and antioxidant agent. The optimized dispersive liquid–liquid microextraction (DLLME) followed by high-performance liquid chromatography–ultra violet detection (HPLC–UV) was used for the analysis of melatonin in human plasma. Influence variables such as volume of extracting (carbon tetrachloride: CCl₄) and dispersing solvents (acetonitrile: ACN), pH and ionic strength, extraction time and centrifugation time were screened in a 2⁶⁻² fractional factorial design (FFD) and then the significant variables were optimized by using a central composite design (CCD). At optimum conditions values of variables set as pH 6.0, 1.5 mL ACN, 140 μL CCl₄, 1.0 min extraction time and 3.0 min centrifugation at 4500 rpm. At optimum conditions method has linear response over 2.0–500.0 ng mL⁻¹ with detection limit of 0.5 ng mL⁻¹ …