Despite recent advances made towards improving the safety and efficacy of lentiviral gene therapies, a large proportion of vector RNA genomes produced are incomplete and thus non-functional. This increases manufacturing costs and must be improved in order to facilitate the widespread clinical implementation of lentiviral gene therapies. Here, we compare three long-read sequencing technologies for their ability to identify sites leading to aberrant lentiviral vector RNA. In order to do so, we develop a bioinformatic analysis and visualisation workflow that facilitates straightforward identification of sites contributing to incomplete RNA. We determine Nanopore direct RNA sequencing to be the most powerful approach in diagnosing issues in vector design. We also show how this approach identifies cryptic splice sites and cryptic polyadenylation sites leading to incomplete RNAs. Using artificial polyadenylation of the RNA, we also identify a multiple hairpin-associated truncations in our lentiviral vectors, which account for the majority of truncated RNA. In summary, Nanopore direct RNA sequencing is a powerful tool for the optimisation of lentiviral vectors, which may help to facilitate the wider use of lentiviral gene therapies.