Antisense oligonucleotide-mediated splicing modulation is an attractive strategy for treating genetic disorders. In 2016, two splice-switching oligonucleotides (SSOs) were approved by the FDA. To date, various types of novel artificial nucleic acids have been developed, and their potential for splicing modulations has been demonstrated. To apply these novel chemistries to SSOs, it is necessary to determine the appropriate design for each artificial nucleic acid, such as the length of the SSO and number of modifications. In this protocol, we focus on SSOs modified with 2′-O,4′-methylene-bridged nucleic acid (2′,4′-BNA)/locked nucleic acid (LNA), which is an artificial nucleic acid that shows extremely high binding affinity to target RNA strands. We describe our typical protocol for the optimization of 2′,4′-BNA-based SSOs.