Melatonin is a natural hormone and with the advancement of age its production declines and thereby may result in some neurological disorders.Exogenous administration of melatonin has been suggested as a neuroprotective agent. Due to its low oral bioavailability, the loading of melatonin in polymeric nanoparticles could be an important tool to effectively use exogenous melatonin. The quantification of the incorporated drug within polymeric nanoparticles is an important step in nanoparticles characterization.An analytical method using high performance liquid chromatography equipped with photodiode array detector(HPLC-PDA)was developed and validated for melatonin determination in poly(lactic acid)nanoparticles obtained by a single emulsion-solvent evaporation technique.The melatonin in vitro release profile also was determined by the HPLC method.Mobile phase consisted of acetonitrile:water(65:35,v/v)pumped at a flow rate of 0.9 mL/min,in the isocratic mode and PDA detector was set at 220 nm.The method was validated in terms of the selectivity,linearity,precision, accuracy,robustness,limits of detection and quantification.Analytical curve was linear over the concentration range of 10–100 μg/mL, and limits of detection and quantification were 25.9 ng/mL and 78.7 ng/mL, respectively. The mean recovery for melatonin was 100.47% (RSD = 1.25%, n = 9). In the intra- and inter-assay,the coefficient of variation was less than 2%.Robustness was proved performing changes in mobile phase, column temperature and flow rate.The method was suitable for the determination of melatonin encapsulation efficiency in poly(lactic acid)nanoparticles and for the evaluation of melatonin in vitro release profile.