During the past five years, the number of single-use bioreactors used in biopharmaceutical research and production has increased tremendously. This increase has been particularly associated with mammalian cell culture processes from small- to medium-scale volumes. Even though nowadays customers can choose from a multitude of 2nd and 3rd generation single-use bioreactors, ranging from mL- up to m3-scale, there is a lack of knowledge of their engineering parameters. Different approaches have been applied to characterization investigations, resulting in an inability to compare different single-use bioreactors with each other and their reusable counterparts, creating an obstacle to a systematic approach to scaling-up the process. This article describes parametric, experimental and computer-based numeric methods for biochemical engineering characterization of single-use bioreactors, which have already been used successfully for the characterization of their reusable counterparts. For the first time, these methods have been evaluated in terms of their practical application.