The invention relates to a method for preparing transmembrane pH-gradient vesicles. This method includes the following steps: a) preparing vesicles made from at least one matrix substance in an aqueous medium having an osmolarity of not more than 200 mOsm/l. The matrix substance is chosen from the group consisting of amphiphilic lipids and amphiphilic block copolymers, b) transferring the vesicles into a basic or acidic buffer having an osmolarity being at least 200 mOsm/l higher than the osmolarity of the aqueous medium of step a) to apply an osmotic shock to the vesicles and to obtain buffer-filled vesicles and c) diluting a mixture of the aqueous medium and the basic or acidic buffer containing the buffer-filled vesicles by adding a neutralizing solution to obtain transmembrane pH-gradient vesicles suspended in a suspension buffer, which differs from the basic or acidic buffer in pH value.