Currently, safety assessment of chemical substances is predominantly based on in vivo animal data. Multiple ethical, scientific, financial and legal considerations and limitations call for the use of alternative testing strategies. The human relevance of alternative testing strategies based on non-animal test methods is usually uncertain. Therefore, there is a need for a validated and accepted framework to assess human relevance of alternative testing methods. A framework to assess human relevance of in vitro models was drafted using an example case study of craniofacial malformations after in utero exposure to triazoles. For this toxicological effect, a (qualitative) Adverse Outcome Pathway (AOP) has been established. While assessing the human relevance for this case, questions were drafted. These questions form the outline of the human relevance framework for the assessment of in vitro methods. The framework starts with an established AOP with sufficient weight of evidence and of which the adverse outcome is relevant to humans. Assessment of human relevance starts with the molecular initiating event, followed by the key events and key event relationships of the pathway. Support for human relevance of the AOP is evaluated by comparing the pathology of human syndromes that have a similar adverse outcome. Lastly, semi-quantitative assessment of human relevance is completed. The combined evidence is scored as ‘strong’, ‘moderate’ or ‘weak’ support of human relevance, based on expert judgement. Overall, the framework is a step forward for application of AOPs and related NAMs in human risk assessment