This study presents a comprehensive profiling of impurities in in vitro transcribed (IVT) mRNA, integrating current technologies with innovative analytical tools. The authors developed highly reproducible and efficient methods, including ion-pair reversed-phase liquid chromatography (IP-RPLC) and capillary gel electrophoresis (CGE), to determine mRNA purity from different suppliers. They also introduced microcapillary electrophoresis (mCE) for high-throughput impurity profiling, achieving analysis times of less than 1.5 minutes per sample. The study revealed that impurities are primarily 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 and native mass spectrometry were used to characterize mRNA and its related product impurities, providing valuable insights into mRNA identity, integrity, and heterogeneity. The findings highlight existing bottlenecks and opportunities for improvement in the analytical characterization of IVT mRNA, contributing to the refinement and streamlining of mRNA production and advancing biotechnological applications.This study presents a comprehensive profiling of impurities in in vitro transcribed (IVT) mRNA, integrating current technologies with innovative analytical tools. The authors developed highly reproducible and efficient methods, including ion-pair reversed-phase liquid chromatography (IP-RPLC) and capillary gel electrophoresis (CGE), to determine mRNA purity from different suppliers. They also introduced microcapillary electrophoresis (mCE) for high-throughput impurity profiling, achieving analysis times of less than 1.5 minutes per sample. The study revealed that impurities are primarily 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 and native mass spectrometry were used to characterize mRNA and its related product impurities, providing valuable insights into mRNA identity, integrity, and heterogeneity. The findings highlight existing bottlenecks and opportunities for improvement in the analytical characterization of IVT mRNA, contributing to the refinement and streamlining of mRNA production and advancing biotechnological applications.