After decades of research, gene therapy products have reached market maturity in recent years. Recombinant adeno-associated viruses (rAAVs) are one of the most promising gene delivery vehicles and are currently under intense scientific investigation. These next-generation medicines remain very challenging when it comes to designing appropriate analytical techniques for quality control. One critical quality attribute is the integrity of ssDNA incorporated in these vectors. The genome is the active compound driving rAAV therapy and therefore requires proper assessment and quality control. Current techniques for rAAV genome characterization include next-generation sequencing, quantitative polymerase chain reaction, analytical ultracentrifugation (AUC), and capillary gel electrophoresis (CGE), yet each of them presents their limitations or lack of user-friendliness. In this work, we demonstrate for the first time the potential of ion pairing-reverse phase-liquid chromatography (IP-RP-LC) to characterize the integrity of rAAV genomes. The obtained results were supported by two orthogonal techniques, AUC and CGE. IP-RP-LC can be performed above DNA melting temperatures, avoiding the detection of secondary DNA isoforms, and does not require the use of dyes due to UV detection. We demonstrate that this technique is suitable for batch comparability, different rAAV serotypes (AAV2 and AAV8), internal vs external (inside vs outside the capsid) DNA analysis, and contaminated samples. Overall, it is exceptionally user-friendly, needs limited sample preparation, has high reproducibility, and permits fractionation for further peak characterization. All of these factors add significant value of IP-RP-LC to the analytical toolbox of rAAV genome assessment.