Reconstitution of membrane proteins into liposomal membranes represents a key technique in enabling functional analysis under well-defined conditions. In this review, we provide a brief introduction to selected methods that have been developed to determine membrane protein orientation after reconstitution in liposomes, including approaches based on proteolytic digestion with proteases, site-specific labeling, fluorescence quenching and activity assays. In addition, we briefly highlight new strategies based on single vesicle analysis to address the problem of sample heterogeneity.