A forced degradation study of a proprietary short interfering RNA (siRNA) molecule most of whose constituent nucleotides have been modified at the 2' position was conducted to assess degradation pathways and stability liabilities. The siRNA was subjected to various conditions as a solid and in solution followed by analysis with reverse-phase ultra-performance liquid chromatography-mass spectrometry. Positional isomers of degradants gave rise to multiple chromatographic peaks with identical masses. In some instances, the exact location of a modification was elucidated, but in most cases although the identity of the nucleotide affected was proposed with a high degree of confidence, its position within the oligonucleotide sequence was not determined. Reaction mechanisms were proposed for all observed major degradants based on reverse-phase ultra-performance liquid chromatography-mass spectrometry data generated in this laboratory and a search of literature sources. This work demonstrates that the chemistry at the 2' position of constituent nucleotides controls degradation pathways of highly modified siRNA molecules under various conditions and that classes of degradants can be predicted with a fair amount of confidence. A table of mass differences is presented that can be used as an aid to making partial structural assignments in oligonucleotide molecules containing similarly modified nucleotides.