Antisense oligonucleotides (AONs) have been actively developed for more than 30 years as a form of molecular medicine and represent promising therapeutic tools for many disorders. Significant progress has been made towards their clinical development in particular for splice switchingSplice switching AONs for the treatment of neuromuscular disorders, such as Duchenne muscular dystrophy (DMD). Many different chemistries of AONs can be used for splice switching modulation, and some of them have now reached regulatory approval. However, despite advances in AON chemistry and design, systemic use of AONs is limited due to poor tissue uptake and sufficient therapeutic efficacy is difficult to achieve. Therefore, there is still a critical need to develop efficient AONs able to target all relevant tissues and international efforts are currently on going to advance new compounds or alternative chemistries with higher therapeutic potential. Here we describe the methods to evaluate the potency of tricyclo-DNA (tcDNA)-based AONs, a constrained class of AONs which displays unique pharmacological properties and unprecedented uptake in many tissues after systemic administration (Goyenvalle, Griffith, Babbs et al., Nat Med 21:270–275, 2015; Goyenvalle, Leumann, Garcia, J Neuromuscul Dis 3:157–167, 2016; Relizani, Griffith, Echevarría et al., Mol Ther Nucleic Acids 8:144–157, 2017; Robin, Griffith, Carter et al., Mol Ther Nucleic Acids 7:81–89, 2017; Relizani, Echevarría, Zarrouki et al., Nucleic Acids Res 50:17–34, 2022). We will focus on the preclinical evaluation of these tcDNA for DMD, specifically targeting the exon 51 of the human dystrophin gene. We will first detail methods to analyse their efficacy both in vitro in human myoblasts and in vivo in the hDMD and mdx52 mouse models and then describe means to evaluate their potential renal toxicity.