Vectors based on adeno-associated virus (AAV) show great promise for safe, efficacious therapeutic gene transfer in extensive pre-clinical data and, recently, in clinical trials. Careful vector design and choice from a range of natural or synthetic pseudotypes allow targeted, efficient, and sustained expression of therapeutic genes. The efficiency of gene delivery can be further enhanced through the use of drug pre-treatment or co-infection with a suitable helper virus. This chapter describes current best practice for AAV production, including complete methods for: (1) efficient generation of vector without the use of helper viruses, simplifying the transition to GMP-grade production for clinical applications; (2) efficient and easily scalable purification of the virus by affinity chromatography, allowing rapid production of highly concentrated, high titre stocks; (3) reliable quantification and assaying of viral stocks, along with short- and long-term storage considerations.