Most vaccines require co-delivery of an adjuvant in order to generate the desired immune responses. However, many currently available adjuvants are non-biodegradable, have limited efficacy, and/or poor safety profile. Thus, new adjuvants, or self-adjuvanting vaccine delivery systems, are required. Here, we proposed a self-adjuvanting delivery system that is fully defined, biodegradable, and non-toxic. The system is produced by conjugation of polyleucine to peptide antigen, followed by self-assembly of the conjugate into nanoparticles. The protocol includes solid-phase peptide synthesis of the vaccine conjugate, purification, self-assembly and physicochemical characterization of the product. Overall, this protocol describes, in detail, the production of a well-defined and effective