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Despite the extensive use of next-generation sequencing of RNA, simultaneous sequencing and quantitative mapping of multiple RNA modifications remain challenging. Herein, we develop MLC-Seq, a mass spectrometry-based direct sequencing method allowing for simultaneously unravelling the RNA sequences and quantitatively mapping different tRNA nucleotide modifications site-specifically. Importantly, MLC-Seq reveals the stoichiometric changes of tRNA modifications upon treatment with the dealkylating enzyme AlkB, and led to the discovery of new nucleotide modifications.