Validation is essential for the translation of newly developed alternative approaches to animal testing into tools and solutions suitable for regulatory applications. Formal approaches to validation have emerged over the past 20 years or so and although they have helped greatly to progress the field, it is essential that the principles and practice underpinning validation continue to evolve to keep pace with scientific progress. The modular approach to validation should be exploited to encourage more innovation and flexibility in study design and to increase efficiency in filling data gaps. With the focus now on integrated approaches to testing and assessment that are based on toxicological knowledge captured as adverse outcome pathways, and which incorporate the latest in vitro and computational methods, validation needs to adapt to ensure it adds value rather than hinders progress. Validation needs to be pursued both at the method level, to characterise the performance of in vitro methods in relation their ability to detect any association of a chemical with a particular pathway or key toxicological event, and at the methodological level, to assess how integrated approaches can predict toxicological endpoints relevant for regulatory decision making. To facilitate this, more emphasis needs to be given to the development of performance standards that can be applied to classes of methods and integrated approaches that provide similar information. Moreover, the challenge of selecting the right reference chemicals to support validation needs to be addressed more systematically, consistently and in a manner that better reflects the state of the science. Above all however, validation requires true partnership between the development and user communities of alternative methods and the appropriate investment of resources.