Recovery of recombinant adenoviruses from infected mammalian cell cultures often requires multiple unit operations such as cell lysis for virus release, microfiltration for clarification, and ultrafiltration for concentration. While development of these multiple unit operations is relatively straightforward, implementation under aseptic conditions in a closed system can be challenging for the production of virus seed at industrial scales. In this study, we have developed a simple, single-step, scaleable process to effectively recover adenoviruses from infected PER.C6 cell cultures for the production of concentrated adenovirus seeds under aseptic conditions. Specifically, hollow fiber tangential flow filtration technology was applied to maximize cell lysis of infected cultures for virus release while simultaneously concentrating the virus to an appropriate level of volume reduction. Hollow fiber filters with small lumen diameter of 0.5 mm were chosen to maximize the wall shear for a highly effective cell lysis and virus release. Cell lysis and virus release were shown to correlate with the exposure time in the hollow fiber cartridge: the shear zone. In most cases, a virus recovery yield of more than 80% and a 15- to 20-fold concentration (or up to 95% volume reduction) was achieved in less than 2 h of processing time. The virus seeds prepared using this process at lab scale and at 300-L scale without clarification have been successfully tested for sterility and potency and used for subsequent infection with consistent virus productivity. This process should enable rapid production of adenovirus seeds with minimal unit operations and high efficiency recovery for adenovirus production at 1000-L scale.