MALDI in combination with high-energy collision-induced dissociation (CID) performed by tandem time-of-flight mass spectrometry (TOF/RTOF) is a relatively new technology for the structural analysis of various classes of biomolecules as e.g., peptides, carbohydrates, glycoconjugate drugs and lipids. Fragmentation mechanisms for these classes of compounds as well as corresponding fragment ion nomenclatures based mainly on data from tandem magnetic sector mass spectrometers are summarized in this article. The major instrumental differences between the present commercially available TOF/RTOFs are compiled (e.g., ion gate, gas-collision cell, type of reflectron, etc.). Whereas peptides have been investigated by MALDI-TOF/RTOF and their CID spectra are well understood, other classes of compounds (e.g., carbohydrates or lipids) are far less well investigated. By comparing data from two different MALDI-TOF/RTOF-instruments, it becomes evident that as they are operated at rather different collision energies for CID (1 versus 20 keV) strong differences in corresponding CID spectra for the same analyte are observed, causing problems with library searches in databases as e.g., abundant peptide side-chain fragmentations mainly occurring in the 8 to 20 keV collision regime are not considered. In contrast, differences in CID spectra of carbohydrates among different TOF/RTOF instruments are less clear-cut, because the required collision energy is spread across a wide range. Especially, carbohydrate cross-ring cleavages require less collision energy in the keV-range than the corresponding peptide side-chain fragmentations. Some of these carbohydrate cross-ring fragmentations are even observed by very low energy CID (< 1 eV fragmentation amplitude). Similar observations can also be made for glycoconjugates (e.g., the drug tylosin A). The lipid class triacylglycerol needs rather high collision energies for dissociating carbon-carbon bonds based upon classical charge-remote fragmentation mechanisms. Comparison of high-energy CID-data of ESI generated triacylglycerol precursors with CID spectra from MALDI generated precursors shows different mechanisms for charge-remote fragmentations. MALDI-TOF/RTOF-instruments operated in the elevated high-energy CID mode exhibit a strong potential in structural analysis of natural and synthetic biomolecules with information often not obtainable by low energy CID.