<p>Here, we describe a practical step-by-step protocol for directional conjugation of monoclonal antibodies to polymersome nanoparticles via the Fc antibody moiety using metal-free click chemistry. This Protocol Extension details procedures for synthesis, quality control and evaluation of nanoparticle–antibody conjugates. The synthesis includes three main stages: (i) a mild oxidation of the glycosyl moiety that is present only on the Fc region of the antibody to produce functional aldehyde groups; (ii) attachment of a heterobifunctional linker, aminooxy-PEG-dibenzocyclooctyne, to the aldehyde groups in the Fc moiety; and (iii) click conjugation of the PEG-linker-modified antibodies to nanoparticles with azide functional groups. This protocol enables covalent surface conjugation of monoclonal antibodies that inherently does not involve the antigen-binding Fab region, thus minimizing the impact of the conjugation on the avidity of antibodies. In contrast, conventional conjugation methods that use amine groups of antibodies for covalent immobilization result in a random antibody orientation that can lead to a loss of the antibody’s binding efficiency. The entire conjugation protocol requires ~18 h. The nanoparticle–antibody conjugates synthesized using this method are expected to display a high molecular specificity toward epitopes of the conjugated antibodies while maintaining the physicochemical properties of the core nanoparticles. The protocol described here does not require any special expertise other than general laboratory training on equipment such as a centrifuge, ultraviolet–visible spectrophotometers, dynamic light scattering, ELISA plate readers and cell culturing.</p>