Asymmetric unilamellar vesicles have been created using five main strategies: i) The inverted emulsion droplet method uses a lipid-coated oil-water interface to generate GUVs. This approach produces GUVs efficiently; however, the size distribution is broad, and residual organic solvents are often present in the hydrophobic core of the bilayer, which may alter the properties of the bilayer and compromise its integrity . ii) Changes in pH can manipulate the protonation state of limited anionic lipids, such as phosphatidylglycerol (PG) and phosphatidic acid (PA), to flip between bilayer leaflets in their uncharged state. This creates an uneven lipid distribution, with accumulation on the more alkaline side of the bilayer . iii) Like cellular systems, carrier molecules with hydrophobic cavities, such as methyl-β-cyclodextrin or lipid transfer proteins , are commonly employed to facilitate the exchange of lipids in the outer leaflet of vesicles. Catalyzed lipid exchange is compatible with a wide range of phospholipids; however, separating the two vesicle populations is needed afterward to isolate the desired asymmetric liposomes . iv) The hemifusion method is used to mix the lipid composition of GUVs with a supported lipid bilayer (SLB) by inducing membrane fusion using calcium ions. In the hemifusion state, the two leaflets are connected through a hemifusion diaphragm, promoting lipid exchange between the outer leaflets via lateral diffusion. Further fusion events are blocked by removing ions with calcium chelation, and a mechanical shear is applied to detach the GUVs from the SLB . v) The enzymatic method can effectively modify lipid composition in specific bilayer leaflets. For instance, sphingomyelin can be hydrolyzed into ceramide and phosphocholine, while PIP5K1C catalyzes the conversion of PI(4)P to PI(4,5)P 2 , as illustrated in Figure . This approach enables selective modification of the outer leaflet of synthetic liposomes without affecting the inner leaflet, as the enzymes used are typically not membrane-permeable. However, its application is limited by enzyme availability and cost, substrate specificity, and the need for precise reaction conditions.