The ‘lipid-coated ice-droplet hydration method’ was applied for the preparation of milliliter volumes of a suspension of giant phospholipid vesicles containing in the inner aqueous vesicle pool in high yield either calcein, α-chymotrypsin, fluorescently labeled bovine serum albumin or dextran (FITC-BSA and FITC-dextran; FITC=fluorescein isothiocyanate). The vesicles had an average diameter of ca. 7–11 μm and contained 20–50% of the desired molecules to be entrapped, the entrapment yield being dependent on the chemical structure of the entrapped molecules and on the details of the vesicle-formation procedure. The ‘lipid-coated ice droplet hydration method’ is a multistep process, based on i) the initial formation of a monodisperse water-in-oil emulsion by microchannel emulsification, followed by ii) emulsion droplet freezing, and iii) surfactant and oil removal, and replacement with bilayer-forming lipids and an aqueous solution. If one aims at applying the method for the entrapment of enzymes, retention of catalytic activity is important to consider. With α-chymotrypsin as first model enzyme to be used with the method, it was shown that high retention of enzymatic activity is possible, and that the entrapped enzyme molecules were able to catalyze the hydrolysis of a membrane-permeable substrate which was added to the vesicles after their formation. Furthermore, one of the critical steps of the method that leads to significant release of the molecules from the water droplets was investigated and optimized by using calcein as fluorescent probe.