Synthetic systems are widely used to unveil the molecular mechanisms of complex cellular events. Artificial membranes are key examples of models employed to address lipid-lipid and lipid-protein interactions. In this work, we developed a new synthetic system that more closely resembles the lysosome - the lysosome-mimicking vesicles (LMVs) - displaying stable acid-to-neutral pH gradient across the membrane. To evaluate the advantages of this synthetic system, we assessed the distinct effects of sphingosine (Sph) accumulation in membrane structure and biophysical properties of standard liposomes (no pH gradient) and in LMVs with lipid composition tuned to mimic physiological- or NPC1-like lysosomes. Ternary 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/Sphingomyelin (SM)/Cholesterol (Chol) mixtures with, respectively, low and high Chol/SM levels were prepared. The effect of Sph on membrane permeability and biophysical properties was evaluated by fluorescence spectroscopy, electrophoretic and dynamic light scattering. The results showed that overall Sph has the ability to cause a shift in vesicle surface charge, increase membrane order and promote a rapid increase in membrane permeability. These effects are enhanced in NPC1- LMVs. The results suggest that lysosomal accumulation of these lipids, as observed under pathological conditions, might significantly affect lysosomal membrane structure and integrity, and therefore contribute to the impairment of cell function.