A rapid, reliable and sensitive LC-MS/MS method for the determination of melatonin in milk was developed and validated. Sample was extracted with dichloromethane and cleaned by passing through Chem Elut solid phase extraction cartridge. The solvent was evaporated to dryness, reconstituted with methanol and analysed by LC-MS/MS on Agilent zorbax Eclipse XDB C-18 rapid resolution analytical column. The analytical procedure was found to be accurate, precise and linear. The method accuracy was 92.2 % (range 90.06-94.58) and the mean precision was 1.55 % and the calibration was linear for 1 to 150 pg mL −1 (R 2 >0.99), the lowest limit of quantification (LLOQ) was 1 pg mL −1. 7-D Melatonin (7-DM) was used as an internal standard. This method was proved to be a promising method for the determination of melatonin for market milk and human milk samples.