This application note describes the validation of the single-use ReadyToProcess WAVE 25 rocking bioreactor system in production of influenza virus from Vero cells in microcarrier-based cultures. A 2 L process, developed using the predecessor WAVE Bioreactor™ 20/50 system, was used as a starting point. Compared with WAVE Bioreactor 20/50, the successor ReadyToProcess WAVE 25 system allows using a smoother rocking motion to reduce shear. As shear stress is a critical parameter in virus production from anchorage-dependent cells, the possibility to increase working volume to 4 L, without increasing shear forces, was investigated. The results show improved culture performance using a smoother rocking motion. To reduce the need for microcarrier preparation prior to use, presterilized and ready-to-use Cytodex Gamma microcarriers were evaluated and compared with Cytodex microcarriers. The results show comparable culture performance between the Cytodex products.