A fundamental feature of eukaryotic cells is the presence of distinct organelles surrounded by lipid bilayers. Assembly and maintenance of the various organellar membranes requires translocation of lipids from one leaflet of the bilayer to the other. Specific membrane proteins, termed lipid flippases, play an essential role in this transport process. We have recently characterized several members of the P4 subfamily of P-type ATPases as prime candidate lipid flippases in the secretory pathway of several eukaryotic cells. Our studies in yeast, plants and mammalian cells uncovered that these pumps serve important functions in vesicular traffic, their activities being required to support vesicle formation in the secretory and endocytic pathways. We are now aiming at determining the mechanism by which these ATPases function in vesicle biogenesis. For this purpose, we are using novel biophysical approaches based on giant vesicles and several advanced bioimaging methods. The limitations and future perspectives of these techniques for the characterization of lipid translocases will be discussed in the light of our recent results.