Abstract: In vitro processes for detecting one or more reactive metabolites that may be formed from a substrate (e.g., a drug or a potential drug candidate) by an enzyme system is disclosed. The substrate is contacted in a mixture with an enzyme system (e.g., with a microsomal drug metabolizing enzyme system, such as a P450 system) to form reactive species (e.g., reactive metabolites), which in the same or a different mixture are contacted with a compound (e.g., glutathione ethyl ester) that reacts with the reactive species to form detectable species (e.g., glutathione ethyl ester conjugates). Preferably, solid phase extraction, high performance liquid chromatography, electrospray ionization, and tandem triple quadropole mass spectrometry are used for detection. The processes may be used in the early stages of a drug discovery program, as well as in other contexts.