Yellow fever (YF) is a life-threatening viral disease endemic in large areas 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 due to limited supply of the current egg-derived vaccine; rare but fatal vaccine adverse effects occurred; and cases were imported to Asia, where the mosquito vector circulates and where local transmission could potentially start. In this work, we investigated the production of YF virus-like particles (VLPs) using suspension-adapted stably-transfected HEK293 cells. In order to develop an intensified process, we combined two strategies: the use of sequential FACS rounds to enrich the stable cell pool in terms of high producers, and the use of perfusion processes. At first, shaken tube experiments revealed that FACS enrichment of the cell pool allowed doubling VLP production, and that in pseudoperfusion cultures (with daily medium exchange) lasting 14 days VLP production increased by 8.3 fold as compared to batch cultures lasting 11 days. When true perfusion cultures were carried out in bioreactors, the use of an inclined cell settler as cell retention device showed operational advantages as compared to an ATF system.