Encapsulation efficiency (EE) of mRNA-based therapeutics and vaccines is defined as the percentage of total mRNA that is efficiently protected by the delivery vehicle from nuclease degradation. As a critical quality attribute, EE must be assessed to ensure that sufficient mRNA evades enzymatic degradation and traverses biological barriers to reach the cellular machinery for translation, without triggering unwanted immune responses caused by free mRNA. In this study, we developed a strategy based on anion exchange chromatography (AEX) to separate lipid nanoparticles (LNPs) and free mRNA based on their charge differences. Carryover issues were mitigated by using a washing step with surfactant, high pH, and high salt concentration. EE was determined by analyzing undiluted samples for free mRNA and measuring total mRNA after LNP disruption using surfactants. The method was successfully applied to the analysis of 30 different mRNA-LNP samples to determine EE. The results were compared to those obtained with the RiboGreen assay, one of the reference methods to assess EE. Our results revealed significant discrepancies between the two techniques that could be explained by the structural information obtained under AEX conditions. Indeed, while the RiboGreen assay provides information on the quantification of mRNA accessible to the fluorescent dye, AEX allows relative quantification of mRNA dissociated from LNPs and information on the presence of surface-localized mRNA as well as transmembrane mRNA. These findings establish AEX as a reliable EE assay, providing information on mRNA distribution within LNPs and advancing the fundamental understanding of LNP structure-function relationships. Based on these features, it offers critical guidance for the rational design of next-generation mRNA therapeutics.