Despite the extensive use of next-generation sequencing (NGS) of RNA, simultaneous direct sequencing and quantitative mapping of multiple RNA nucleotide modifications remains challenging. Mass spectrometry (MS)-based sequencing can directly sequence all RNA modifications without being limited to specific ones, but it requires a perfect MS ladder that few tRNAs can provide. Here, we describe an MS ladder complementation sequencing approach (MLC-Seq) that circumvents the perfect ladder requirement, allowing de novo MS sequencing of full-length heterogeneous cellular tRNAs with multiple nucleotide modifications at single-nucleotide precision. Unlike NGS-based methods, which lose RNA modification information, MLC-Seq preserves RNA sequence diversity and modification information, revealing new detailed stoichiometric tRNA modification profiles and their changes upon treatment with the dealkylating enzyme AlkB. It can also be combined with reference sequences to provide quantitative analysis of diverse tRNAs and modifications in total tRNA samples. MLC-Seq enables systematic, quantitative, and site-specific mapping of RNA modifications, revealing the truly complete informational content of tRNA.