Singlet oxygen (¹O₂) is known to be produced by a number of enzymes as well as by UV or visible light in the presence of a sensitizer. It also can be formed during the dimerization reactions of peroxyl radicals in lipid peroxidation, via the Russell mechanism. Singlet oxygen shows considerable reactivity toward electron-rich organic molecules, leading to the formation of endoperoxides, dioxetanes or allylic hydroperoxides. Therefore, biomolecules such as lipids, proteins and DNA are important targets of ¹O₂. Lipid double bonds react with ¹O₂ by the ene reaction_, yielding hydro-peroxides. In proteins, only cysteine, histidine, methionine, tryptophan, and tyrosine react significantly with ¹O₂ at neutral pH. For example, it was shown that ¹O₂ oxidation generates dioxetanes and/or hydroperoxides within the side chain residues of aromatic amino acids. Moreover, in DNA, guanine is the only nucleic acid component that exhibits significant reactivity toward ¹O₂ at neutral pH. One important product of this reaction is 8-oxo-7,8-dihydro-2’-deoxyguanosine, a mutagenic lesion. The peroxides formed by ¹O₂ oxidation and their decomposition products have been shown to give rise to further damage, leading to deleterious biological effects such as aging, neurodegenerative disease, atherosclerotic lesions, mutagenesis, and carcinogenesis. Recently, the development of new techniques, using ¹⁸O-labeled compounds and HPLC coupled to tandem mass spectrometry (HPLC-MS/MS), has provided insights to elucidate the mechanisms by which peroxides can increase initial damage in biological systems. In this work, we report some aspects of ¹O₂ oxidative damage to biomolecules and recent developments in the understanding of this type of damage, using techniques such as HPLC-MS/MS.