The interpretation of the numerous sequence variants of unknown biological and clinical significance (UV for “unclassified variant”) found in genetic screenings or patients represents a major challenge in the molecular diagnosis of genetic disease, including cancer susceptibility. A fraction of UVs may be deleterious because they affect mRNA splicing. The most direct approach to determine whether disease-causing mutations are associated with splicing is to perform a reverse transcription PCR (RT-PCR) analysis on RNA from the relevant tissues of affected individuals. However, tissue samples are often not available because the expression of these genes are tissue specific (for example only in brain or heart) or patients are too far from laboratory. An alternative approach is to test the effects of the splicing mutations using minigenes. Here, we describe a functional splicing assay based on a minigene construct that assesses the impact of sequence variants on splicing. A genomic segment encompassing the variant sequence of interest along with flanking intronic sequences is PCR-amplified from patients genomic DNA and is cloned into a minigene vector. After transient transfection into cultured cells, the splicing patterns of the transcripts generated from the wild-type and from the mutated constructs are compared by reverse transcription-PCR analysis and sequencing. With classical minigenes is possible have difficulties to test mutations because is difficult clone the fragment that contains this mutation in the minigene. Many times the DNA fragment containing restriction sites incompatible with restriction sites of minigene, or the ligation reaction between the fragment and minigene is hardly for the size of the fragment. For bypass this problem we cloned a Gateway cassette that allow fast and easy cloning. Whit this assay we tested 16 various splicing mutation in different genes (NF1, CFTR, AIP, COQ6, STK11) with conclusive result