Melatonin is a lipophilic hormone with limited water solubility, which is physiologically synthesized in the human body by the pineal gland. It has a pleiotropic effect on the main physiological functions, the cell cycle, and biochemical mechanisms with circadian characteristics. Therapeutic use is primarily related to sleep disorders such as insomnia. Nowadays, research is focused on the development of innovative pharmaceutical forms of melatonin. The production of nanosuspensions represents one of the strategies for the formulation of poorly soluble drugs with the aim of increasing their solubility, dissolution rate and bioavailability. However, the main limitation is the long-term instability of nanosuspensions and the tendency of particles to aggregate. Freeze drying, i.e. lyophilisation, proved to be one of the methods of developing solid forms of nanosuspensions with increased physical stability. The aim of this work was to examine the possibility of lyophilisation of melatonin nanosuspension for the purpose of developing a dry form using glucose, mannitol and trehalose (0.5-2.5%, w/v) as cryoprotectants, and to examine their influence on the short-term and long-term stability of the developed lyophilisates by measuring size, polydispersity index and zeta-potential of nanocrystals in reconstituted suspensions. Nanosuspension of melatonin (0.143%, w/v) was prepared by the method of wet milling with the addition of polysorbate 80 (0.286%, w/v) as a surfactant, in a weight ratio of 1:2. The particle size within the initial nanosuspension was 176.4 ± 3.3 nm with a polydispersity index of 0.141 ± 0.013. Lyophilisation of the melatonin nanosuspension in the presence of mannitol resulted in a lyophilisate volume equal to the volume of the suspension before lyophilisation. In the case of trehalose, a partial collapse was observed, and in the case of glucose, a complete collapse of the structure of the lyophilisate was observed. Mannitol and trehalose at concentrations from 0.5 to 2.5 % (w/v) ensured satisfactory preservation of the physical properties of melatonin nanocrystals after lyophilisation, in contrast to glucose. Increasing the concentration of mannitol and trehalose in the lyophilised nanosuspension partially increased the success of preserving the size and size distribution of nanocrystals. During storage at 4 °C for a period of 60 days, with mannitol and trehalose (2.5 %, w/v) as cryoprotectants, the appearance of the lyophilisate and the physical properties of the nanocrystals after reconstitution were successfully preserved, thus confirming their cryoprotective effect in the process of lyophilisation of melatonin nanosuspensions.