With the rapid growth of synthetic messenger RNA (mRNA)-based therapeutics and vaccines, the development of analytical tools for characterization of long, complex RNAs has become essential. Tandem liquid chromatography-mass spectrometry (LC-MS/MS) permits direct assessment of the mRNA primary sequence and modifications thereof without conversion to cDNA or amplification. It relies upon digestion of mRNA with site-specific endoribonucleases to generate pools of short oligonucleotides that are then amenable to MS-based sequence analysis. Herein, we showed that the uridine-specific human endoribonuclease hRNase 4 improves mRNA sequence coverage in comparison with the benchmark RNase T1 by producing a larger population of uniquely mappable cleavage products. We deployed hRNase 4 to the analysis of mRNAs fully substituted with 1-methylpseudouridine (m1Ψ) or 5-methoxyuridine (mo5U), as well as mRNAs selectively depleted of uridine–two key strategies to reduce synthetic mRNA immunogenicity. To further illustrate the power of hRNase 4 to interrogate mRNA sequence, we demonstrated that hRNase 4 provides near 60% sequence coverage of a fully m1Ψ -modified 4,187 nt mRNA encoding the primary sequence of the BNT162b2 COVID-19 vaccine. Lastly, we leverage hRNase 4 to identify the presence of a 7-methylguanosine 5′-triphosphate 2′-O-methyladenosine (m7GpppAm) cap structure on an IVT mRNA.