The present chapter focuses on how the use of [18O]-labeled endoperoxides and hydroperoxides can be applied to study mechanistic aspects related to singlet molecular oxygen (1O2) generation and reactions in biological system. Identification of 1O2, which was achieved using different approaches: [18O]-labeled molecular oxygen, a clean naphthalene derivative [18O]-labeled thermolabile endoperoxide as a source of [18O]-labeled 1O2, chemical trapping of [18O]-1O2 with anthracene derivatives and liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The peroxidation reactions of the main cellular targets including nucleic acids, proteins and unsaturated lipids have received major attention during the last two decades. We highlighted some key mechanistic aspects related to [18O]-peroxides decomposition to alkoxyl or peroxyl radicals. As a biochemical application this chapter focused on the recombination reactions involving lipid-derived [18O]-labeled peroxyl radicals that can be formed on different lipid species giving rise to [18O]-labeled 1O2 through the mechanism proposed by Russell. A relevant major topic deals with the search for the molecular signature of the 1O2 formation in targeted biomolecules within cells. It may be anticipated that labeled peroxides and [18O]-labeled 1O2 in association with a sensitive mass spectrometric method such as LC-ESI-MS/MS should constitute powerful tools for this purpose.