The ability to verify the sequence of a nucleic acid-based therapeutic is an essential step in the drug development process. The challenge associated with sequence identification increases with the length and nuclease resistance of the nucleic acid molecule, the latter being an important attribute of therapeutic oligonucleotides. We describe methods for the sequence determination of Spiegelmers, which are enantiomers of naturally occurring RNA with high resistance to enzymatic degradation. Spiegelmer sequencing is effected by affixing a label or hapten to the 5′-end of the oligonucleotide and chemically degrading the molecule in a controlled fashion to generate fragments that are then resolved and identified using liquid chromatography-mass spectrometry. The Spiegelmer sequence is then derived from these fragments. Examples are shown for two different Spiegelmers (NOX-E36 and NOX-A12), and the specificity of the method is shown using a NOX-E36 mismatch control
