The rapid development of mRNA-based therapeutics, especially post-COVID-19, has necessitated the precise characterization of mRNA quality attributes, including sequence integrity. Ion-pairing reversed-phase liquid chromatography (IP-RPLC) has been widely accepted as a reference method for the characterization of small oligonucleotides. Some studies have already investigated the use of IP-RPLC for RNA, but no systematic approach has been developed to assess the impact of ion-pairing agents (IPAs) on the separation of large RNA molecules. This study addresses this gap by investigating the potential of IP-RPLC for the separation and characterization of large RNA molecules, with a specific focus on optimizing the use of IPAs to enhance retention and selectivity. Thirteen different IPAs, varying in hydrophobicity, were systematically tested using a supermacroporous polymeric (divinylbenzene) column with a very broad pore size range under various conditions, including different temperatures, pH, and IPA concentrations. The results demonstrate that moderately hydrophobic IPAs provide superior resolution for RNA species up to 6000 nucleotides. An optimized combination of 100 mM butylammonium acetate and 50 mM tripropylammonium acetate achieved the best overall separation, significantly improving resolution by 35% compared to individual IPAs. The study also identifies optimal conditions for RNA separation, including a mobile phase pH of 7.0, acetonitrile as the organic solvent, and a column temperature of 65 °C. In a second step, a solution to increase the retention of small nucleotides and thereby separate nucleic acids ranging from 1 to 6000 nucleotides allowing to characterize IVT-mRNA differing in length and study their integrity and fragmentation or monitor the presence of in-process impurities (nucleotides) was investigated by combining two different LC columns. These findings enhance the analytical toolbox for evaluating the critical quality attributes of RNA, supporting the development of reliable and efficient RNA-based therapeutics.