Attaching targeting ligands on the surface of self-assembled drug delivery systems is the key request for a controlled transport of the drug to a desired location. Most commonly, the amphiphilic molecules (block-copolymers, lipids etc.) are therefore pre-functionalized before the self-assembly takes place. However, this strategy cannot be applied, if it interferes with the self-assembly process, if the introduced functional groups react with loaded cargo or if natural carriers like extracellular vesicles should be functionalized. Here, we present the site-specific coupling of antibodies to the surface of amino group-terminated liposomes via bio-orthogonal copper-free click chemistry after liposome formation. The present primary amino groups were functionalized with a linker carrying a strained alkyne group for a bio-orthogonal strain-promoted alkyne–azide cycloaddition (SPAAC) reaction where Cu(I) as a catalyst can be avoided. Antibodies were site-specifically functionalized with azide moieties along the Fc region to avoid interference with the antigen binding sites. The liposome surface functionalization reaction was optimized by precisely analyzing the number of available functional groups (both amine and alkyne), which often represents a challenge for self-assembled systems. By finally confirming the successful antibody coupling, we provide a facile and robust functionalization strategy, which can be applied to a wide range of self-assembled systems and desired targeting antibodies maintaining physiological conditions throughout the procedure.