In this study, we introduce triglyceride-based nanodroplets and their use in studying peptide and protein lipid interaction as a new biomimetic lipid model, complementary to the lipid bilayers and monolayers formed at the water/air interfaces (Langmuir monolayers). Specifically, stable lipid nanodroplets, i. e. nanoemulsions of lipid droplets (LDs) composed of trioleoylglycerol core covered by a monolayer of sphingomyelin (SM) and cholesterol (Chol), have been prepared by our recently developed combined reverse-phase evaporation/ultrasonication method. By the employment of asymmetric-flow field-flow fractionation, light scattering, electro-kinetic mobility measurements, and transmission electron microscopy, the prepared LDs were examined for lipid composition, ζ-potential, gyration and hydrodynamic radius, shape, and temporal stability and compared to extruded SM/Chol large unilamellar vesicles (LUVs). To the best of our knowledge, this is the first in-depth characterization of LDs covered by a SM/Chol monolayer. LDs and LUVs with theoretical SM/Chol molar ratios of 1/1 and 4/1 were further investigated for the orientational order of their interfacial water molecules using a second harmonic scattering technique, and for interactions with the SM-binding and Chol-binding pore-forming toxins equinatoxin II and perfringolysin O, respectively. The surface characteristics and binding of peptide and protein to the LDs SM/Chol monolayers were similar to those for the SM/Chol bilayers of the LUVs in terms of their surface structures. For that reason, we propose that LDs enable to study pore formation process. In conclusion, our findings suggest that such SM/Chol/TOG nanoparticles with the required lipid compositions can serve as experimental models for monolayer membrane to provide a system that imitates the natural lipid droplets.