The use of Integrated Testing Strategies (ITS) enables the combination of diverse types of chemical and toxicological data for hazard identification and characterisation. In November 2008, the European Partnership for Alternative Approaches to Animal Testing (EPAA), together with ECVAM, held a workshop on ‘Overcoming Barriers to Validation of Non-animal Partial Replacement Methods / Integrated Testing Strategies’ in Ispra, Italy to discuss to what extent current ECVAM approaches to validation can be used to evaluate partial-replacement in vitro test methods (i.e. as potential ITS components) and ITS themselves. The main conclusion of these discussions was that formal validation was only considered necessary for regulatory purposes (e.g. replacement of a test guideline) and that current ECVAM approaches to validation should be adapted to accommodate such test methods (Kinsner-Ovaskainen et al, 2009a). With these conclusions in mind, a follow-up EPAA-ECVAM workshop was held in October 2009 to discuss to what extent existing validation principles are applicable to validation of ITS test methods and to develop a draft approach for validation of such test methods and/or overall ITS for regulatory purposes. This report summarises the workshop discussions which started with a review of current validation methodologies and the presentation of two case studies (skin sensitisation and acute toxicity) before covering the definition of ITS components and ITS themselves, their validation and their regulatory acceptance. The following conclusions/recommendations were made: 1) the validation of a partial replacement test method (for application as part of a testing strategy) should be differentiated from the validation of an in vitro test method for application as a stand-alone replacement, especially with regard to its predictive capacity. In the former case, the predictive capacity of the whole testing strategy (rather than of the individual test methods) would be more important, in particular if the individual test methods have a high biological relevance; 2) an ITS allowing for flexible and ad hoc approaches cannot be validated, whereas the validation of a clearly defined ITS would be feasible, although practically quite difficult; 3) test method developers should be encouraged to develop and submit to ECVAM not only full replacement test methods, but also partial replacement methods to be placed in a testing strategy. The added value of formal validation of testing strategies and the requirements needed in view of regulatory acceptance of the data requires further informed discussion within the EPAA forum on the basis of case studies provided by industry.