Adeno-associated viral vectors (AAV) are one of the most efficient engineered tools for delivering genetic material into host cells. The commercialization of AAV-based drugs goes hand in hand with the need to increase manufacturing capacities and to develop appropriate quality controls. In particular, accurate methods to assess the level of residual DNA in AAV vector stocks are needed, considering the potential risk of co-transferring oncogenic or immunogenic sequences with the therapeutic vectors. Our laboratory has developed an assay based on high-throughput sequencing (HTS) to exhaustively identify and quantify DNA species in recombinant AAV batches. Compiled with a computational analysis of the single nucleotide variants (SNV), Single-Stranded Virus Sequencing (SSV-Seq) also provides information regarding rAAV genome identity. In this study, we show that the PCR amplification of regions with high GC content and including mononucleotide stretches can be biased during the DNA library preparation, leading to drops in the sequencing coverage along the AAV vector genome. To circumvent this problem, we have developed a PCR-free protocol, named SSV-Seq 2.0, that is optimized for the sequencing of rAAV genomes that contain sequences with a high percentage of GC and homopolymers, such as the CAG promoter. HTS-based assays are indispensable to provide accurate data to the regulatory agencies regarding nucleic acids content in AAV vector batches and to improve the safety and efficacy of these viral vectors.