Abstract: Gene therapy, particularly employing adeno-associated virus (AAV) vectors, has emerged as a revolutionary frontier in medical treatments. As the field progresses towards clinical applications, ensuring the potency of these therapeutics is essential. Potency assays play a crucial role, assessing the specific ability of the product to achieve desired therapeutic outcomes; specifically, the translation of the delivered DNA into mRNA. Equally as important as the assay itself is the ability for it to be robust and reproducible. Thus, automation of RNA extraction, quantification, normalization, and qPCR for these assays greatly improves our ability to achieve that. Our goal was to move from a low throughput manual method to an automated96 well format with minimal touchpoints. Automation of RNA extraction is the first pivotal step in our innovation. By leveraging the Eppendorf epMotion robotic systems along with Beckman Coulter SPRI bead technology, we have optimized and standardized the extraction process, significantly reducing manual errors and variability. This not only expedites the assay workflow but also enhances the reliability of results, laying the foundation for more precise clinical assessments. Our initial work compared the SPRI bead method to column-based extraction methods in 6 well culture plates. With similar quantity and quality of RNA between the two extraction methods we were able to move to 96 well culture plates for increased throughput. This format was easily adaptable to our automation platform. After RNA purification, quantification and normalization of RNA samples are performed on our automated platform. Normalization steps are automated to guarantee consistent sample inputs across the assay, mitigating potential biases and enhancing the overall reliability of potency assessments. The final component of our automated RNA potency assay involves quantitative polymerase chain reaction (qPCR), as an in-vitro potency readout for AAV gene therapy. This optimized and automated RNA potency assay greatly improves our throughput and robustness in the lab. This innovation not only addresses the challenges associated with traditional potency assays but also sets the stage for more efficient and reliable clinical development. The assay gives our laboratory the ability to deliver treatments from discovery to patients as fast as possible.