Giant unilamellar vesicles (GUVs) with a semi-permeable nature are prerequisites for constructing synthetic cells. Here we engineer semi-permeable GUVs by the inclusion of DOTAP lipid in vesicles. Diffusion of molecules of different charge and size across GUVs are reported. Control over size-selective permeability is demonstrated by modulating the DOTAP lipid composition in different lipid systems without reconstituting membrane proteins. Such semi-permeable GUVs have immense applications for constructing synthetic cells.