Chitosan-based microspheres have been extensively studied as mucoadhesive and controlled release drug delivery systems. One of the mostly used methods for their preparation is the spray-drying method, which allows for the creation of microparticles of desired physical-chemical properties. The stability of microparticles not only depends on the properties of the active pharmaceutical ingredient and excipients as microparticle constituents, but also on the particle-forming process during the spray drying, which should be taken into consideration when developing a stable product. In this study, melatonin-loaded chitosan and poloxamer/chitosan microspheres were prepared using the previously optimised spray-drying method. The aim of this study was to test the stability of the prepared microspheres in relation to their composition and storage conditions. Microspheres were stored at the temperature of 4-8°C, and at the temperature of 25°C and relative humidity of 60%. The stability of microspheres was evaluated by monitoring the microsphere mean geometric diameter, size distribution and surface charge over six-month period and determining the content of the drug entrapped after 6-month and 2-year period of storage. Microspheres were characterised by mean diameter ranging between 2.5 and 2.8 μm, zeta-potential ranging between 29 and 34mV and high melatonin entrapment efficiency ( 99.5 %). The drug content in the chitosan and poloxamer/chitosan microspheres prepared was 33.2 ± 0.2% and 29.4 ± 1.2%, respectively. Melatonin-loaded chitosan microspheres preserved their physical properties during the six-month storage at the temperature of 4-8°C, while a mild decrease in zeta-potential was observed at a temperature of 25°C and the relative humidity of 60%. Melatonin-loaded poloxamer/chitosan microspheres preserved their physical properties during the six-month storage regardless of the storage conditions. Microsphere stability in terms of drug content depended on microsphere composition rather than storage conditions. Drug content in poloxamer/chitosan microspheres remained unchanged after 6-months and 2-years of storage at temperature of 4-8°C as well as at temperature of 25°C and relative humidity of 60%, while in case of chitosan microspheres, a slight decrease in drug content was observed after 2 years of storage at the same conditions. The results obtained within this investigation revealed higher stability of melatonin-loaded poloxamer/chitosan microspheres in relation to chitosan microspheres with the same drug entrapped.