The use of antisense oligonucleotides to modulate splicing patterns has gained increasing attention as a therapeutic platform and, hence, the mechanisms of splice-switching oligonucleotides are of interest. Cells expressing luciferase pre-mRNA interrupted by an aberrantly spliced β-globin intron, HeLa pLuc705, were used to monitor the splice-switching activity of modified oligonucleotides by detection of the expression of functional luciferase. It was observed that phosphorothioate 2′-O-methyl RNA oligonucleotides containing locked nucleic acid monomers provide outstanding splice-switching activity. However, similar oligonucleotides with several mismatches do not impede splice-switching activity which indicates a risk for off-target effects. The splice-switching activity is abolished when mismatches are introduced at several positions with locked nucleic acid monomers suggesting that it is the locked nucleic acid monomers that give rise to low mismatch discrimination to target pre-mRNA. The results highlight the importance of rational sequence design to allow for high efficiency with simultaneous high mismatch discrimination for splice-switching oligonucleotides and suggest that splice-switching activity is tunable by utilizing locked nucleic acid monomers.