This study investigates the relationship between the structure of oligonucleotides (ONs) and their separation in hydrophilic interaction chromatography (HILIC), focusing on phosphorothioate (PS)-modified ONs. PS modifications introduce chirality, leading to the formation of diastereomers. The research identifies higher-order structures (HOSs) as the primary factor influencing diastereomer separation in HILIC. By analyzing ON folding and using mass spectrometry and HILIC, the study demonstrates how chromatographic parameters such as column temperature, pore size, stationary phase, and mobile-phase ionic strength can enhance or suppress diastereomer separation. The findings suggest that HILIC can effectively characterize PS-containing ONs, enabling the monitoring of diastereomer distributions in full-length siRNAs. The study also highlights the importance of ON structure in diastereomer separation, showing that more rigid HOSs lead to better resolution. Additionally, the impact of sugar modifications, PS number and location, and duplexes on diastereomer separation is explored. The research underscores the role of hydrogen-bonding interactions in HILIC separation and provides strategies to suppress diastereomer separation when needed. Overall, the study contributes to a better understanding of ON structure and its influence on HILIC-based characterization.This study investigates the relationship between the structure of oligonucleotides (ONs) and their separation in hydrophilic interaction chromatography (HILIC), focusing on phosphorothioate (PS)-modified ONs. PS modifications introduce chirality, leading to the formation of diastereomers. The research identifies higher-order structures (HOSs) as the primary factor influencing diastereomer separation in HILIC. By analyzing ON folding and using mass spectrometry and HILIC, the study demonstrates how chromatographic parameters such as column temperature, pore size, stationary phase, and mobile-phase ionic strength can enhance or suppress diastereomer separation. The findings suggest that HILIC can effectively characterize PS-containing ONs, enabling the monitoring of diastereomer distributions in full-length siRNAs. The study also highlights the importance of ON structure in diastereomer separation, showing that more rigid HOSs lead to better resolution. Additionally, the impact of sugar modifications, PS number and location, and duplexes on diastereomer separation is explored. The research underscores the role of hydrogen-bonding interactions in HILIC separation and provides strategies to suppress diastereomer separation when needed. Overall, the study contributes to a better understanding of ON structure and its influence on HILIC-based characterization.