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Natural nucleic acids are the molecules of life. They contain the genetic information of every organism and are involved in many biological processes. Additionally, they were involved for the development of life on earth. All nucleic acids contain natural modified nucleotides, which are incorporated in complex processes and are of tremendous importance for regulation and accuracy of transcription and translation in every organism. Over 120 different modifications were detected in all natural nucleic acids and the number is still increasing. tRNA is the most heavily modified nucleic acid and it additionally contains the broadest structural variety of modifications. So far, the function and role of single tRNA modifications has been investigated. Natural modified nucleosides were only rarely investigated in a context based manner. To overcome this limitation, we developed a method which allows quantification of in principle all tRNA modifications in parallel. This is similar to proteomics and metabolomics research in which the complete proteome or metabolome of a cell is quantified in parallel, respectively.