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The ATP-binding cassette subfamily A member 4 gene (ABCA4)-associated retinopathy, Stargardt disease, is the most common monogenic inherited retinal disease. Given the pathogenicity of numerous ABCA4 variants is yet to be examined and a significant proportion (more than 15%) of ABCA4 variants are categorized as splice variants in silico, we therefore established a fibroblast-based splice assay to analyze ABCA4 variants in an Australian Stargardt disease cohort and characterize the pathogenic mechanisms of ABCA4 variants. A cohort of 67 patients clinically diagnosed with Stargardt disease was recruited. Genomic DNA was analysed using a commercial panel for ABCA4 variant detection and the consequences of ABCA4 variants were predicted in silico. Dermal fibroblasts were propagated from skin biopsies, total RNA was extracted and the ABCA4 transcript was amplified by RT-PCR. Our analysis identified a total of 67 unique alleles carrying 74 unique variants. The most prevalent splice-affecting complex allele c.[5461-10T > C; 5603A > T] was carried by 10% of patients in a compound heterozygous state. ABCA4 transcripts from exon 13 to exon 50 were readily detected in fibroblasts. In this region, aberrant splicing was evident in 10 out of 57 variant transcripts (18%), carried by 19 patients (28%). Patient-derived fibroblasts provide a feasible platform for identification of ABCA4 splice variants located within exons 13–50. Experimental evidence of aberrant splicing contributes to the pathogenic classification for ABCA4 variants. Moreover, identification of variants that affect splicing processes provides opportunities for intervention, in particular antisense oligonucleotide-mediated splice correction.