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Purpose Pathogenic variants in PRPF31 are a leading cause of autosomal dominant retinitis pigmentosa (RP). Despite advances in next-generation sequencing, many patients remain undiagnosed, due to variants of uncertain significance (VUS). These include non-coding variants affecting splicing, which are challenging to interpret. This study uses reverse-transcription polymerase chain reaction (RT-PCR) combined with Oxford Nanopore Technology (ONT) long-read sequencing to functionally characterise non-coding PRPF31 variants.Methods Six individuals with RP and non-coding PRPF31 variants were investigated, including known pathogenic variants (c.527+3A>G and c.528-39_531del) and VUS (c.856-8T>G, c.946-3C>G, c.239-3C>G, c.855+3G>C). SpliceAI and Pangolin were used for in silico splice prediction. RT-PCR was performed on RNA extracted from peripheral blood mononuclear cells, followed by ONT sequencing of full-length PRPF31 transcripts. Results were compared with computational predictions.Results ONT sequencing enabled full-length transcript analysis in a single assay, demonstrating both dominant and low-level splicing events. This included multiple splicing alterations arising from individual non-coding PRPF31 variants, as well as ubiquitous low-level mis-splicing occurring across the gene, reflecting the inherent complexity of physiological splicing. Notably, two variants, c.946-3C>G and c.527+3A>G, demonstrated clear splicing defects on ONT analysis, despite having insignificant SpliceAI scores, highlighting the limitations of in silico predictive tools.Conclusion This study demonstrates utility of ONT-based transcript analysis for functional validation of non-coding PRPF31 VUS, resolving uncertainties over pathogenicity and enabling molecular diagnoses. Findings provide proof-of-principle for integrating ONT-sequencing into clinical diagnostics to aid variant interpretation, benefiting unsolved inherited retinal disease patient cohorts globally. This work also offers insights into complex splicing alterations driven by non-coding variants in PRPF31, advancing understanding of disease mechanisms and informing future therapies.