With the increased number of identified nucleotide sequence variations in genes, the current challenge is to classify them as disease causing or neutral. These variants of unknown clinical significance can alter multiple processes, from gene transcription to RNA splicing or protein function. Using an approach combining several in silico tools, we identified some exons presenting weaker splicing motifs than other exons in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene. These exons exhibit higher rates of basal skipping than exons harboring no identifiable weak splicing signals using minigene assays. We then screened 19 described mutations in three different exons, and identified exon-skipping substitutions. These substitutions induced higher skipping levels in exons having one or more weak splicing motifs. Indeed, this level remained under 2% for exons with strong splicing motifs and could reach 40% for exons having at least one weak motif. Further analysis revealed a functional exon splicing enhancer within exon 3 that was associated with the SR protein SF2/ASF and whose disruption induced exon skipping. Exon skipping was confirmed in vivo in two nasal epithelial cell brushing samples. Our approach, which point out exons with some splicing signals weaknesses, will help spot splicing mutations of clinical relevance.