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Virus-like particles (VLPs) are increasingly used for vaccine development and drug delivery. Assembly of VLPs from purified monomers in a chemically defined reaction is advantageous compared to in vivo assembly, because it avoids encapsidation of host-derived components and enables loading with added cargoes. This review provides an overview of ex cella VLP production methods focusing on capsid protein production, factors that impact the in vitro assembly, and approaches to characterize in vitro VLPs. The uses of in vitro produced VLPs as vaccines and for therapeutic delivery are also reported.