Significance Membrane proteins (MPs), encoded by ∼30% of the coding genes, play vital roles in numerous physiological processes. MPs are targets of more than half of the FDA-approved drugs. High-resolution structural studies of functional membrane proteins under near-physiological conditions are required to provide an in-depth mechanistic understanding and to facilitate drug discovery. To embed the proteins into liposomes represents a strategy to mimic native membrane conditions. Here we present a convenient workflow for cryo-EM analysis of liposome-embedded MPs using the prototypal protein AcrB. Our method sets the foundation for future investigation of MPs in the presence of electrochemical gradients and for the understanding of the interdependence of integral or peripheral MPs and various membrane properties.