Oligonucleotide therapeutics, which include antisense oligonucleotides, small interfering RNAs, and aptamers, show promising clinical outcomes for disease indications. Multi types of oligonucleotide, approved by FDA, have been successfully applied for the treatment of different types of diseases. The need for robust, accurate, and efficient ways of detecting such oligonucleotides and their metabolites in complex bodily fluids is crucially important for pharmacokinetic, pharmacodynamic, and exposure-response studies. Bioanalysis strategy of oligonucleotides is constantly evolving primarily driven by drug development needs and regulatory requirements, which also along with the increased diversity of the oligo¬nucleotide structure and the advancement of sample preparation/extraction technologies. Though LBA assay is the predominant bioanalytical tool for large molecular, with recent advances in small molecule bioanalytical technologies, more and more small molecule bioanalytical technologies are used to support the bioanalysis of oligonucleotide therapeutics along with unique advantages, such as hybridization-based HPLC-Fluorescence, LC–MS/MS and LC-HRMS assays. To support preclinical and clinical studies, each method with different advantages and disadvantages has been used for different study purposes for a period of time.