Sanger sequencing is the standard preclinical method used for sequence verification of AAV transfer vector cloning constructs and eventual packaged viral genomes. While Sanger sequencing does provide high quality data in a relatively short turnaround time, the data analysis requires manual evaluation of sequencing reads, rendering this approach incredibly low-throughput. Additionally, Sanger sequencing only provides 1-2X coverage, which results in low confidence single nucleotide polymorphism (SNP) and indel calling. It is imperative for preclinical gene therapy labs and core facilities to sequence both the transfer vector plasmids that go into viral production and the eventual packaged genomes to ensure no recombinations or mutations have occurred, and to validate that the genomes were not truncated during capsid packaging.This protocol outlines a simple, fast, and inexpensive methodology geared toward preclinical environments for sequencing packaged AAV genomes via NGS, using paired-end Tn5 tagmentation-based library preparation. This protocol was optimized for use on the commonly available illumina MiSeq instrument, but other instruments would also work and should be chosen based on the number of samples and depth required (iSeq 100, MiniSeq, MiSeq, NextSeq, etc.).