This application guide highlights the need for highly sensitive analytical assays to support preclinical evaluation of antisense oligonucleotide (ASO) therapeutics. It provides detailed protocols for a range of in vitro and in vivo methods, including 2D cell culture ASO dosing, immunocytochemistry (ICC), Anti-PS and Anti-MOE ELISA assays, 3D spheroid dosing, 3D cell culture ICC, in vivo ASO dosing, and tissue immunohistochemistry (IHC). In addition, the guide offers an overview of oligonucleotide drug modalities, their key therapeutic disease areas, and links to antibodies targeting clinically relevant oligonucleotide modifications.