Animal based research needs to strictly adhere to the 3R principle (replace, refine, reduce), not only for the ethical justification of the use of animals in science, but also to ensure the highest quality of data due to standardization. With the implementation of the EU Directive 2010/63 within national law it is mandatory to perform a 'severity classification of procedures on the basis of estimated levels of pain, suffering, distress and lasting harm that is inflicted on the animals'. However, scientifically sound scales as well as broadly accepted and applicable model-specific measures to grade this in routine work are lacking. This deficit in the research landscape impacts ethical considerations as well as the quality of animal based research data. In addition, the discrepancy of current regulations and scientific knowledge momentarily hinders biomedical research that still requires testing hypotheses in animals. Therefore, the current Special Issue 'Severity Assessment' contains reports from relevant research projects and networks that were implemented to