Despite their limited cargo capacity (<5 kb), adeno-associated viral (AAV) vectors remain the gold standard for in vivo delivery of therapeutic genes. Dual AAV vectors have emerged as a valuable tool for delivering large therapeutic genes and CRISPR tools to overcome this limitation. Here we provide a detailed protocol for the design, production and evaluation of dual AAV vectors. We offer guidelines for selecting a suitable dual AAV strategy, designing and cloning the genes to be delivered, and conducting in vitro evaluations of expression efficiency. In addition, we detail the production of dual AAVs and their assessment in human cellular models, such as induced pluripotent stem cell-derived retinal organoids. Finally, we outline the administration of dual AAVs via different routes in mice and the assessment of transgene-derived RNA and protein expression in various tissues. Overall, the instructions in this Protocol will aid in the efficient in vivo delivery of large DNA fragments using dual AAVs. This Protocol is adaptable to a wide range of model organisms as well as to human organoid cultures and, depending on the application, can be completed in 15–44 weeks.