Publisher Summary In well-characterized chemical systems, singlet oxygen can be produced by both photochemical and nonphotochemical processes, and its intermediacy can be quantitatively determined. Considerable recent interest has focused on the question of the intermediacy of singlet molecular oxygen (1O2) in biological processes, formed either as a result of natural processes or under the effect of exogenous agents, such as photosensitizers. In biological systems (which are often inhomogeneous), demonstration of its intermediacy in the oxidation of target species is not easy. The main problem is that singlet oxygen is likely to be accompanied by other reactive oxygen species, such as superoxide ion (O2-), hydroxyl radical (OH・), and alkoxy and peroxy radicals. The reactions of these species may compete with or be confused with those of singlet oxygen. The four main methods for the detection of singlet oxygen in a reacting system are to compare observed products with those known to be produced by singlet oxygen, to determine the change in the amount of reaction or effect produced on adding substances that modify the lifetime of singlet oxygen, to measure the luminescence of singlet oxygen, or to produce singlet oxygen independently in the system and compare its reactions with those observed under the test conditions. All of these methods have difficulties; the greatest degree of certainty is obtained by using as many independent techniques as possible in combination. This chapter discusses few of the available methods.