The use of plasmids is critical in the gene therapy viral vector workflow: plasmids are widely used for creation of the viral vector construct, along with helper and packaging plasmids for final product creation. While the construct insert is universally sequenced during initial design, sequencing of the backbone of the plasmid is not frequently assessed. Sequence errors within the plasmid backbone, helper and packaging plasmids can have major impacts in downstream packaging and viral vector production. Traditional Sanger approaches can be used for fast and frequent characterization, however limitations within the technology necessitate many sequencing reactions to span the full plasmid, with a known reference sequence. Traditional Next Generation Sequencing (NGS) approaches can overcome the need for multiple reactions and a reference sequence but are historically slower and cost-prohibitive. Here we describe a fast and cost-effective sequencing approach for sequencing circular plasmid DNA to generate a highly accurate and annotated full-length consensus sequences without the need for primer design or a known reference sequence. This long-read sequencing method is comparable in cost and time to Sanger sequencing but unlike Sanger sequencing, overcomes common issues within hard to sequence regions like long nucleotide repeats or inverted terminal repeat (ITR) domains of adeno-associated viruses (AAVs). When used effectively, it can be harnessed to characterize gene therapy viral vector plasmid and associated packaging plasmids required throughout the gene therapy development life cycle without delay, ultimately accelerating the viral vector design and development process.