Abstract: Therapeutic oligonucleotides have become as a new drug class. They are synthetic DNA or RNA that typically between 15 and 50 nucleotide units long. In the pharmacokinetic studies of therapeutic oligonucleotides, the classical method was enzyme-linked immunosorbent assay (ELISA). However, the development of ELISA method was time-consuming and expensive, and has become the bottleneck in the new drug research and development. The technical issues associated with liquid chromatography-mass spectrometry (LC/MS) method in the bioanalysis of oligonucleotides included: limited ionization efficiency, severe matrix effect, incompatibility of chromatographic condition and MS detection, and low sample preparation recovery. However, the development of an LC/MS method was fast， and this method was selective. This review summarized the development and applications of LC/MS method for the quantitation of therapeutic oligonucleotides and characterization of their metabolites in biological samples described in the literatures over the past 15 years.