Therapeutic oligonucleotides (ONs) are short strands of DNA or RNA used for the treatment of diseases that cannot be efficiently targeted by other drug modalities. Antisense oligonucleotides (ASOs) represent an important class with the largest number of approved drugs. ASOs function as geneexpression inhibitors or splicing modulators, aiming to modulate protein expression. ASOs are typically synthesized by solid-phase supported synthesis (SPSS) and the sequential additions of single nucleotides to a growing chain, employing four reactions per nucleotide: de-blocking (detritylation), coupling, oxidation/sulfurization and capping. During synthesis, side-reactions, reaction failures and degradation reactions can occur. This commonly results in a full-length product (FLP) comprising a considerable number of closely-related impurities, including shorter and longer sequences (shortmers and longmers, respectively). Moreover, to improve the pharmacokinetic properties of ONs, chemical modifications are often introduced, such as 2'-substituted riboses, methylated bases, or sulfurization of the phosphate group. The latter results in phosphorothioated (PS) groups, which significantly reduce enzymatic (nuclease) degradation in the body compared to phosphodiester ONs, but also increases sample complexity as each PS introduces a chiral center, leading to 2 n diastereomers for an ON with n PS groups. Additionally, to aid targeted drug-delivery, the ON can be covalently conjugated with a ligand to increase cellular uptake e.g. a carbohydrate moiety, such as N-acetylgalactosamine (GalNAc), which facilitates receptor-mediated uptake of ONs specifically to hepatocytes in the liver. As of 2025, there are at least nine candidate ONs in clinical trials that exploit GalNAc conjugation. Clearly, the molecular complexity of current ONs poses a significant analytical challenge.