Conventional vaccines either use attenuated pathogens (live vaccines) or inactivated pathogens, such vaccines can be expensive, difficult to deploy, require a ‘cold chain’, may have side effects and require multiple doses for effective immunisation. Recent advances in the development of technology to delivery biologically active molecules in vivo have utilised nanoparticles for the in vivo delivery of drugs, nucleic acids, proteins and peptides; nanotechnology has a promising potential as a new platform technology for vaccine delivery. Nanoparticles allow controlled release of proteins and increase the vaccine stability with the possibility of eliminating cold chain storage. Freeze-drying of protein-loaded nanoparticles can be used to improve both the short and long-term stability of vaccine components. The purpose of this review is to provide an overview of the freeze-drying process itself with the aim of identifying current procedures and parameters that can impact the stability of the protein-loaded nanoparticles during and after freeze-drying. It also discusses the formulation strategies, nanoparticle stability and the importance of excipients during the freeze-drying process. This review will provide direction towards the successful integration of freeze-drying into the development of protein-based nanovaccines to further enhance the application of this emerging vaccine technology platform.