In the adeno-associated viral vector (rAAV) system, single-stranded transgenic cargo is flanked by two 145nt T-shaped structures consisting of three internal, self-annealed inverted repeats. These structures, termed inverted terminal repeats (ITRs), are the only genomic element of the parental virus retained in the recombinant vector as they are required for vector genome replication, translocation into pre-assembled vector particles, and enhance extrachromosomal stability of the exogenous DNA in transduced cells. In spite of these important roles, the underlying mechanisms and their relation to ITR sequence and structure remain poorly understood. As capsid bioengineering continues to improve the efficacy of specific applications and tissue targets, elucidating the nature of the ITRs stands to benefit the platform as a whole, including both manufacturing and (pre)clinical applications. Unfortunately, although they are presumably stable in their physiological context, plasmid-encoded ITRs are susceptible to mutations and rearrangements, likely due to their high guanine/cytosine content and proclivity for secondary structure. Furthermore, these same features hinder our ability to quantitatively assess the nature and extent of ITR disrepair. The lack of analytical insight into ITR integrity negatively affects basic vectorological research as well as clinical and preclinical studies. We will present our progress towards establishing novel analytical techniques for robust and accurate analysis of ITR integrity in rAAV plasmid constructs. This work will greatly facilitate ITR research and form the foundation of novel quality control (QC) standards that will help enhance the clinical impact of rAAV vectors in gene therapy applications.