Yellow fever (YF) is a life-threatening viral disease endemic in parts of Africa and Latin America. Although there is a very efficacious vaccine since the 1930s, YF still causes 29,000-60,000 annual deaths. During recent YF outbreaks there were issues of vaccine shortage of the current egg-derived vaccine; rare but fatal vaccine adverse effects occurred; and cases were imported to Asia, where the circulating mosquito vector could potentially start local transmission. Here we investigated the production of YF virus-like particles (VLPs) using stably transfected HEK293 cells. Process intensification was achieved by combining sequential FACS (fluorescence-activated cell sorting) rounds to enrich the stable cell pool in terms of high producers and the use of perfusion processes. At shaken-tube scale, FACS enrichment of cells allowed doubling VLP production, and pseudoperfusion cultivation (with daily medium exchange) further increased VLP production by 9.3-fold as compared to batch operation mode. At perfusion bioreactor scale, the use of an inclined settler as cell retention device showed operational advantages over an ATF system. A one-step steric exclusion chromatography purification allowed significant removal of impurities and is a promising technique for future integration of upstream and downstream operations. Characterization by different techniques confirmed the identity and 3D-structure of the purified VLPs.