Flow-following sensor technology offers a method to collect information on flow patterns and local velocities in pilot- and industrial scale reactors, which are practically inaccessible to many measurement techniques. Such data is highly valuable for scale-up of bioprocesses, as well as validation of bioreactor CFD simulations. Flow-following sensors were applied in a pilot-scale (2 m³ filled volume) bubble column fermentor, showing that axially resolved data can be acquired under heterogeneous bubbly flow conditions with high gas holdup. Next the use of the collected data for validation of CFD simulations of the pilot-scale reactor is explored, discriminating between models utilizing different interphase interaction models. The CFD simulation was found capable of capturing the velocity profile and circulation behavior, but full validation was found to be challenging. When simulating virtual sensors via Lagrangian particle tracking, differences are observed in terms of particle distribution and sensitivity to particle density between experimental and simulated data, indicating further development of representative CFD simulations is required.