We report on the successful encapsulation of hydrophobic substrates into the hydrophobic shell of poly(butadiene)-b-poly(ethylene oxide) vesicles in water and straightforward characterization methods. The hydrophobic fluorescent dye Nile Red was embedded into unilamellar vesicles of PB130-b-PEO66 prepared via different methods leading to different average hydrodynamic radii and distributions but a common hydrophobic shell thickness of ∼16 nm as determined by transmission electron microscopy (TEM). The combination of cryogenic TEM and fluorescence microscopy studies shows that the self-assembled structure remains unchanged when the hydrophobic dye is incorporated within the vesicle shell. Furthermore, highly fluorescent quantum dots with an average diameter of d = 5.7 ± 0.6 nm as determined by TEM were selected as hydrophobic model substrates and successfully enclosed into the vesicles, as evidenced by fluorescence correlation spectroscopy (FCS) measurements in combination with dynamic light scattering (DL...