Messenger RNA (mRNA) based drugs represent a new prospective class of products and there is currently significant demand for the development of new and improved analytical methods for the characterization of mRNA therapeutics. The aim of this work was to develop a mass spectrometry-based workflow covering sample preparation, LC-MS analysis and data processing in order to characterize mRNA therapeutics Partial mRNA digestion was performed using immobilized RNase T1 on magnetic beads. mRNA samples were diluted to 0.5mg/mL in digestion buffer, 2.5 µL of the immobilized T1 beads were added. The sample was incubated at 37°C with continuous agitation for 5 minutes. The reaction was stopped using a magnet to completely removes magnetic beads, followed by centrifugation. Chromatographic separation of was performed using a Thermo Scientific™ DNAPac RP column and a Thermo Scientific™ Vanquish™ UHPLC system, at a column temperature of 50°C and flow rate of 250 μL/min. Negative electrospray ionization was performed using a Thermo Scientific™ Orbitrap Exploris™ 240 mass spectrometer. Data analysis was performed with Thermo Scientific™ BioPharma Finder™ 4.1 software. Preliminary results demonstrate the identification of digestion fragments ranging from 2 to 50 bases in length with the chromatographic elution order from the column correlating to digestion fragment length. Additionally, the chromatographic separation conditions demonstrate resolution of isobaric and isomeric compounds with capability to identify sequence isomers based on their MS2 fragmentation patterns. Confidence scores for digestion fragment identification are provided by the data processing software based on identified fragment m/z values as well as by comparison to predicted fragment intensities and expected isotopic patterns. Using this approach, sequence mapping coverage greater than 90% was obtained for each of the mRNA samples analyzed.