Splice-altering variants are suggested to be responsible for part of the missing heritability of inherited retinal diseases (IRDs). The interpretation of these variants is challenging as functional evidence is required to validate pathogenicity. We explored the diagnostic value of a targeted long-read cDNA sequencing (lrcDNA-seq) approach to investigate IRD-associated splicing defects. For each affected individual, RNA was isolated from blood, and for each candidate gene, cDNA amplicons, spanning the complete open reading frame or multiple exons, were generated and subjected to long-read sequencing. We validated our approach by assessing previously described pathogenic splice-altering variants in IRD-associated genes. Next, we investigated six genetically unexplained affected individuals, each carrying pathogenic variant(s) in NMNAT1. In two probands, we provided functional validation for previously identified variants of uncertain significance present on the second allele. In four other subjects, lrcDNA-seq revealed the partial inclusion of an SVA_F retrotransposon in the NMNAT1 mRNA, predicted to introduce a premature stop codon. We showed that targeted lrcDNA-seq is effective in characterizing splice defects and in identifying novel splice-altering variants and uncovered the IRD genetic basis for six previously unexplained subjects. We believe that the implementation of this technique has the potential to contribute to an increased diagnostic rate of IRDs.