Antisense-mediated splicing modulation of premessenger RNA represents a novel therapeutic strategy for several types of pathologies such as genetic disorders, cancers, and infectious diseases. Antisense oligonucleotides designed to bind to specific mRNA molecules have been actively developed for more than 20 years as a form of molecular medicine to modulate splicing patterns or inhibit protein translation. More recently, small nuclear RNA such as U7 or U1 small nuclear RNA have been used to carry antisense sequences, offering the advantage of long-term effect when delivered to cells using viral vectors. We have previously demonstrated the therapeutic potential of U7snRNA targeting dystrophin mRNA as a treatment for Duchenne muscular dystrophy. In particular, we showed that bifunctional U7 snRNAs harboring silencer motifs induce complete skipping of exon 51, and thus restore dystrophin expression in DMD patients cells to near wild-type levels. These new constructs are very promising for the optimization of therapeutic exon skipping for DMD, but also offer powerful and versatile tools to modulate pre-mRNA splicing in a wide range of applications. Here, we outline the design of these U7snRNA constructs to achieve efficient exon-skipping and describe methods to evaluate the efficacy of such U7snRNA constructs in vitro using the dystrophin gene as an example.