Abstract <p>Flavoprotein oxidases have found many biotechnological applications. For identifying and improving their characteristics, it is essential to have reliable and robust assay methodology available. The methodologies used to monitor their activity seem to be scattered in the literature and seem often selected based on convenience. Due to the diversity of reactions catalyzed by flavoprotein oxidases, it is virtually impossible to recommend a single activity assay. A literature analysis of 60 recent papers describing flavoprotein oxidases revealed that continuous spectrophotometric assays, in particular colorimetric assays, are the preferred choice, as they are facile, scalable and allow for better interpretation of data than discontinuous assays. Colorimetric assays typically rely on the extinction coefficient of a monitored chromogenic product, which can be highly variable depending on the experimental conditions. Therefore, it is important to determine the extinction coefficient under the specific experimental conditions used, rather than taking it directly from the literature. To provide a guideline and assist in standardization, this review describes the most commonly utilized activity assays for flavoprotein oxidases, along with their respective merits and limitations.</p> Graphical Abstract <p></p> Key points <p>• <i>Researchers should be more aware of limitations of activity assays.</i></p> <p>• <i>Extinction coefficients should be determined for the appropriate experimental setup.</i></p> <p>• <i>New robust activity assays are desired.</i></p>