Cholesterol is an essential sterol in cell membranes that regulates organization and fluidity. This biomolecule has been identified as one of the critical factors in the internalization process of various viruses in human cells. Therefore, understanding these mechanisms is crucial for a deeper comprehension of viral pathogenicity in the search for practical therapeutic approaches against viral diseases. The biochemical and biophysical processes related to these diseases are highly complex. For this reason, studying model systems capable of mimicking the interaction of lipid membranes with cholesterol and proteins is fundamental. In this work, we propose to study the structural and elastic changes in mono-, bi-, and tridimensional lipid systems composed of dipalmitoylphosphatidylcholine (PC) with varying amounts of cholesterol in the presence and absence of the S protein (Spike) and its receptor-binding domain (RBD) from SARS-CoV-2. To characterize these systems, we used both experimental and theoretical approaches such as Langmuir trough, atomic force microscopy (AFM), small-angle X-ray scattering (SAXS), electrochemical methods, and molecular dynamics (MD) simulations. With the interpretation of all results obtained in this work, it was possible to propose a structural model of the membrane in the presence of cholesterol and the interaction with the Spike protein and RBD. The behavior of the adsorption isotherm and SAXS data, together with the results provided by MD simulations, led us to conclude that cholesterol in PC monolayers promotes local alterations, inducing the formation of more rigid membrane regions. More importantly, cholesterol plays a crucial role in facilitating the allocation of SARS-CoV-2 proteins in lipid systems. This is especially true for the Spike protein, which displayed a non-ACE2 mediated stable binding to the lipid membrane with high internalization.