As small molecule drugs for Duchenne muscular dystrophy (DMD), antisense oligonucleotides (AONs) have been shown to restore the disrupted reading frame of DMD transcripts by inducing specific exon skipping. This allows the synthesis of largely functional dystrophin proteins and potential conversion of severe DMD into milder Becker muscular dystrophy (BMD) phenotypes. We have previously described 37 exon-internal AONs that induce skipping of 14 DMD exons in human control myotube cultures. Here, we report 77 new AONs, effectively targeting an additional 21 exons. Of the 114 AONs thus far tested, 72 (67%) were effective. AON design initially was based on a partial overlap with predicted open secondary structures in the target RNA. We have analyzed various AON and target exon parameters in retrospect. Interestingly, we observed significantly higher SF2/ASF, SC35, and SRp40 values (as predicted by ESEfinder) for effective AONs when compared with ineffective AONs. In addition, the distance to the 3' splice site was significantly smaller for effective AONs. No other significant correlations were observed. Our results suggest that effective exon-internal AONs primarily act by blocking SR binding sites (which often correspond to open structures) and that ESEfinder may be used to refine AON design for DMD and other genes.