Here, we describe the synthesis of novel ESI-cleavable Tandem Nucleic acid mass Tag-peptide nucleic acid conjugates and their analysis by ESI-MS/MS. We demonstrate a novel mass tag cleavage method in which source voltages in the electrospray ionisation source are used to cleave an ESI-cleavable linker, by a collision-based process, releasing the mass tag from the oligonucleotide during ionization. This method allows for direct analysis of assay solutions without requiring complex workups to cleave and isolate tags. In principle, this cleavage method would also allow in-line separation, by capillary electrophoresis for example, of labelled nucleic acids with direct spraying of the separated material into the ion source where tag cleavage would take place automatically.