The Langmuir technique was applied for the first time to compare the layers obtained by spreading lipid liquid-crystalline nanoparticles monoolein 1-oleoyl-rac-glycerol (GMO)/Pluronic F108 cubosomes with the monolayers obtained by mixing the same components in chloroform at the air-water interface. The differences in the monolayer behavior and in the acting intermolecular forces were examined. The similarity of the isotherms obtained for the mixed components system and the cubosome-derived layer proved the disintegration of cubosomes into a single monolayer upon contact with the air-water interface. Despite the low Pluronic F108 content in both types of layers, a strong structural role of this stabilizer was also demonstrated. Cubosome-derived systems supported on hydrophilic mica substrates were prepared either using the combined Langmuir-Blodgett and Langmuir-Schaefer technique or via direct adsorption from the solution. The topographies of the obtained layers were studied by atomic force microscopy (AFM). Images obtained in the air mode revealed the disintegration of cubosomes and the formation of large crystallized structures of the polymer, while AFM imaging performed in water confirmed the presence of intact cubosomes on the surface of mica. We proved that the original structure of cubosomes remains on one condition: the films must not dry out; therefore, the aqueous environment must be preserved. This new approach provides an explanation in the ongoing discussion of what happens to lipid nanoparticles with or without cargo when they come into contact with an interface.