Background: A hormone made by the pineal gland (tiny organ near the center of the brain). Melatonin helps control the body's sleep cycle, and is an antioxidant. It is also made in the laboratory and sold as a supplement. Objectives: A rapid and sensitive analytical method based on liquid chromatography coupled to tandem mass spectrometry detection with ESI positive mode (Turbo spray) was developed for the determination of Melatonin in human plasma using D4-Melatonin as the internal standard (IS). Materials and Methods: A Solid Phase Extraction (SPE) was performed using acetonitrile. The analyte and IS were subjected to chromatographic analysis on an Eclipse XDB C18 (100 mm, 4.6 mm, 3.5 μm) using ACN:0.1% FA in water (80:20) as the mobile phase at a flow rate of 0.600 ml/min. An AB Sciex API 4000 Triple Quad LC/MS was operated in the multiple reaction monitoring modes. The precursor-to-product ion transitions as 232.80/173.9 (m/z) (Melatonin) and 237.10/177.90 (m/z) (D4-Melatonin, IS) were used for quantitation. Results: The results were linear over the studied range (52 to 5176pg/mL) and the total analysis time for each chromatograph was 1.81 ± 0.01 min. The Between run & within run Precision was range between 2.7 and 8.5 %., and the accuracy was within 117.42%. Conclusion: This method can be used for the pharmacokinetics study of melatonin in human plasma.