RNA modifications play crucial roles in regulating cellular processes, but comprehensive mapping of the human RNome still remains limited by technological challenges. Mass spectrometry is a valuable tool to analyse RNA modifications and complementing sequencing-based analysis. Current MS-based oligonucleotide workflows have limited sensitivity, requiring micrograms of RNA inputs and thus hindering studies on native RNAs. Additionally, environmentally toxic ion-pairing reagents are often required. There is also a demand for more specific nucleases and systematic studies on different nucleases. Here, we report a highly sensitive, broadly applicable oligonucleotide-MS workflow that enables analysis of nanogram-scale RNA hydrolysates. We systematically benchmarked the substrate specificity of three nucleases: RNase T1, RNase 4, and Colicin E5 under varying conditions. We developed a nano-flow hydrophilic interaction liquid chromatography (HILIC) setup compatible with common MS buffers and coupled this with high-resolution MS. Using modified NucleicAcidSearchEngine (NASE), we confidently assigned RNA hydrolysates with diverse 3’-end chemistries. Furthermore, we demonstrate that RNase 4 and Colicin E5 efficiently cleave modified RNAs including pseudouridine-containing transcripts, enabling high sequence coverages. Using this workflow, we successfully mapped modifications in 25 ng of native yeast tRNAPhe and verified the sequence of 250 ng of synthetic mRNA. Overall, our method provides a sensitive, high-resolution platform for shotgun RNomics, facilitating comprehensive analysis of RNA modifications in limited biological samples and advancing efforts toward complete epitranscriptomic mapping.