With the rapid growth of synthetic messenger RNA (mRNA)-based therapeutics and vaccines, the development of analytical tools for characterization of mRNA has become essential. Tandem liquid chromatography-mass spectrometry (LC-MS/MS) permits direct assessment of the mRNA primary sequence and modifications, including mRNA 5′ end capping. Herein, we show that the uridine-specific human endoribonuclease RNase 4 (hRNase4) improves mRNA sequence coverage in comparison with the benchmark, RNase T1 by producing a population of longer and uniquely mappable cleavage products. In addition, 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. Lastly, we developed a simple and flexible workflow to selectively isolate and analyze structural features of the 5′ end of an mRNA by means of DNA probe-directed enrichment with hRNase4. hRNase 4 increases the precision of RNA cleavage, reducing product heterogeneity, while providing comparable estimates of capped products and their intermediaries relative to the widely used RNase H. Collectively, this study highlights the power of hRNase 4 to interrogate mRNA sequence, identity, and modifications by LC–MS/MS.