This study presents a comprehensive profiling of impurities in in vitro-transcribed (IVT) mRNA, evaluating current technologies and advanced analytical techniques. The research aims to improve the analytical characterization of IVT mRNA, which is crucial for the development of mRNA-based therapies and vaccines. The study integrates current technologies with innovative analytical tools to determine the purity of mRNA from different suppliers and assess the impact of impurities on mRNA function. The study developed highly reproducible and efficient ion-pair reversed-phase liquid chromatography (IP-RPLC) and capillary gel electrophoresis (CGE) methods for mRNA purity analysis. Additionally, microcapillary electrophoresis (mCE) was introduced for high-throughput mRNA impurity profiling. The findings revealed that impurities are mainly attributed to mRNA variants with different poly(A) tail lengths due to aborted additions or partial hydrolysis and the presence of double-stranded mRNA (dsRNA) byproducts, particularly the dsRNA 3'-loop back form. Mass photometry (MP) and native mass spectrometry (MS) were used to characterize mRNA and its related impurities. MP enabled the determination of the number of nucleotides of different mRNAs with high accuracy and the detection of their size variants, providing valuable information on mRNA identity and integrity. Native MS provided insights into mRNA intact mass, heterogeneity, and important sequence features such as poly(A) tail length and distribution. The study highlights the existing bottlenecks and opportunities for improvement in the analytical characterization of IVT mRNA, contributing to the refinement and streamlining of mRNA production. The results demonstrate the importance of monitoring mRNA impurities, particularly those in the prepeak region, which may compromise mRNA stability. Postpeak impurities were linked to various dsRNA conformations, underscoring the need to monitor their levels. The study also emphasizes the potential of mCE as a promising alternative for mRNA purity assessment, offering similar separation efficiency, data quality, and shorter run times compared to conventional CGE. MP technology was shown to provide valuable additional information, particularly for the detection of noncovalent species. Native MS played a crucial role in monitoring impurities in the postpeak region with sufficient resolution to reflect the different lengths of the poly(A) tail. Overall, the study demonstrates the capacity of these analytical methods to characterize mRNA and its related impurities, highlighting the importance of developing robust technologies for assessing mRNA purity and characterizing impurities, particularly their biological effects. This work represents a pivotal statement in advancing the quality control and assessment of mRNA products, with far-reaching implications for pharmaceutical science and patient care.This study presents a comprehensive profiling of impurities in in vitro-transcribed (IVT) mRNA, evaluating current technologies and advanced analytical techniques. The research aims to improve the analytical characterization of IVT mRNA, which is crucial for the development of mRNA-based therapies and vaccines. The study integrates current technologies with innovative analytical tools to determine the purity of mRNA from different suppliers and assess the impact of impurities on mRNA function. The study developed highly reproducible and efficient ion-pair reversed-phase liquid chromatography (IP-RPLC) and capillary gel electrophoresis (CGE) methods for mRNA purity analysis. Additionally, microcapillary electrophoresis (mCE) was introduced for high-throughput mRNA impurity profiling. The findings revealed that impurities are mainly attributed to mRNA variants with different poly(A) tail lengths due to aborted additions or partial hydrolysis and the presence of double-stranded mRNA (dsRNA) byproducts, particularly the dsRNA 3'-loop back form. Mass photometry (MP) and native mass spectrometry (MS) were used to characterize mRNA and its related impurities. MP enabled the determination of the number of nucleotides of different mRNAs with high accuracy and the detection of their size variants, providing valuable information on mRNA identity and integrity. Native MS provided insights into mRNA intact mass, heterogeneity, and important sequence features such as poly(A) tail length and distribution. The study highlights the existing bottlenecks and opportunities for improvement in the analytical characterization of IVT mRNA, contributing to the refinement and streamlining of mRNA production. The results demonstrate the importance of monitoring mRNA impurities, particularly those in the prepeak region, which may compromise mRNA stability. Postpeak impurities were linked to various dsRNA conformations, underscoring the need to monitor their levels. The study also emphasizes the potential of mCE as a promising alternative for mRNA purity assessment, offering similar separation efficiency, data quality, and shorter run times compared to conventional CGE. MP technology was shown to provide valuable additional information, particularly for the detection of noncovalent species. Native MS played a crucial role in monitoring impurities in the postpeak region with sufficient resolution to reflect the different lengths of the poly(A) tail. Overall, the study demonstrates the capacity of these analytical methods to characterize mRNA and its related impurities, highlighting the importance of developing robust technologies for assessing mRNA purity and characterizing impurities, particularly their biological effects. This work represents a pivotal statement in advancing the quality control and assessment of mRNA products, with far-reaching implications for pharmaceutical science and patient care.