Endosomal escape is a critical challenge in the development of lipid nanoparticle (LNP)-mediated nucleic acid therapies, particularly for mRNA vaccines. LNPs are effective delivery systems for nucleic acids, but their ability to release mRNA from endosomes into the cytosol remains inefficient, limiting therapeutic efficacy. The mechanisms of endosomal escape are not well understood, and current methods for studying this process are limited and inconsistent. This review summarizes the current understanding of endosomal escape mechanisms, the challenges in studying this process, and the need for more robust, quantitative methods to improve LNP-based therapies. LNPs internalize into cells via endocytosis, typically through clathrin-independent mechanisms such as macropinocytosis. Once inside, they are transported to early endosomes, which mature into late endosomes and eventually lysosomes. Efficient delivery requires the release of mRNA-LNPs before lysosomal degradation. However, most mRNA-LNPs are either degraded or recycled, with only a small fraction escaping into the cytosol. The exact endosomal compartment from which escape occurs is still debated, with some studies suggesting early or late endosomes, or a hybrid compartment. Two main theories explain endosomal escape: one involves ionizable lipids interacting with anionic lipids in the endosomal membrane, leading to membrane disruption and release of the payload. The other, the "proton sponge effect," involves the buffering capacity of ionizable lipids, leading to proton influx, chloride accumulation, and endosomal swelling and bursting. These mechanisms are not fully understood, and studies show that factors such as lipid composition, endosomal pH, and cell type influence escape efficiency. Current methods to study endosomal escape include direct imaging of payloads and indirect analysis using endosomal damage indicators or genetic manipulation. However, these methods have limitations, including low resolution and difficulty in detecting low fluorescence signals. Advanced techniques such as single-molecule localization microscopy and membrane-based studies are being explored to better understand the process. Improving endosomal escape is crucial for enhancing the efficiency of LNP-based therapies. Strategies include modifying lipid formulations, using small molecules to induce endosomal damage, or enhancing endosomal retention through genetic manipulation. Additionally, exploring alternative delivery systems that bypass endocytosis, such as fusogenic liposomes or cell-penetrating peptides, may offer new avenues for improving therapeutic outcomes. Despite progress, challenges remain in understanding the exact mechanisms and developing robust methods to enhance endosomal escape for more effective nucleic acid delivery.Endosomal escape is a critical challenge in the development of lipid nanoparticle (LNP)-mediated nucleic acid therapies, particularly for mRNA vaccines. LNPs are effective delivery systems for nucleic acids, but their ability to release mRNA from endosomes into the cytosol remains inefficient, limiting therapeutic efficacy. The mechanisms of endosomal escape are not well understood, and current methods for studying this process are limited and inconsistent. This review summarizes the current understanding of endosomal escape mechanisms, the challenges in studying this process, and the need for more robust, quantitative methods to improve LNP-based therapies. LNPs internalize into cells via endocytosis, typically through clathrin-independent mechanisms such as macropinocytosis. Once inside, they are transported to early endosomes, which mature into late endosomes and eventually lysosomes. Efficient delivery requires the release of mRNA-LNPs before lysosomal degradation. However, most mRNA-LNPs are either degraded or recycled, with only a small fraction escaping into the cytosol. The exact endosomal compartment from which escape occurs is still debated, with some studies suggesting early or late endosomes, or a hybrid compartment. Two main theories explain endosomal escape: one involves ionizable lipids interacting with anionic lipids in the endosomal membrane, leading to membrane disruption and release of the payload. The other, the "proton sponge effect," involves the buffering capacity of ionizable lipids, leading to proton influx, chloride accumulation, and endosomal swelling and bursting. These mechanisms are not fully understood, and studies show that factors such as lipid composition, endosomal pH, and cell type influence escape efficiency. Current methods to study endosomal escape include direct imaging of payloads and indirect analysis using endosomal damage indicators or genetic manipulation. However, these methods have limitations, including low resolution and difficulty in detecting low fluorescence signals. Advanced techniques such as single-molecule localization microscopy and membrane-based studies are being explored to better understand the process. Improving endosomal escape is crucial for enhancing the efficiency of LNP-based therapies. Strategies include modifying lipid formulations, using small molecules to induce endosomal damage, or enhancing endosomal retention through genetic manipulation. Additionally, exploring alternative delivery systems that bypass endocytosis, such as fusogenic liposomes or cell-penetrating peptides, may offer new avenues for improving therapeutic outcomes. Despite progress, challenges remain in understanding the exact mechanisms and developing robust methods to enhance endosomal escape for more effective nucleic acid delivery.