In parallel with the growing interest in nucleic acid-based therapies and the increasing number of oligonucleotide (ON) approvals in recent years, there is a demand for analytical solutions to address the challenges inherent to ON analysis and to improve the characterization of these relatively small, but highly complex drugs. In this thesis, liquid chromatography techniques such as ion-pairing reversed-phase liquid chromatography (IP-RPLC) and hydrophilic interaction chromatography (HILIC) have been investigated. Some of the key challenges in the analysis of ONs were studied and a focus was placed on understanding the fundamentals of the analysis of ONs to develop innovative chromatography-based techniques for ONs.