The characterization of the metabolic pathways of new chemical entities with a special emphasis on detecting potentially reactive metabolites is increasingly being performed early in the drug discovery process. In the present study, the preliminary in vitro metabolic routes of a series of novel 2-substituted benzothiophene-containing discovery molecules were determined in fresh and cryopreserved hepatocyte suspensions. The objectives of this investigation were: (1) to use systematic LC/MS and LC/MS/MS analyses to provide a preliminary characterization of the in vitro metabolism of these compounds, with a particular focus on metabolites potentially arising from reactive intermediates, and (2) to identify potential lead molecules not associated with such metabolic pathways. This benzothiophene-containing series of compounds was characterized by the formation of five metabolites, at least two of which (dihydrodiol formation and glutathione adduct of the dihydrohydroxyl) were indicative of the formation of a reactive arene oxide intermediate. Tandem mass spectral analysis of the metabolites formed from a variety of structurally similar compounds demonstrated this reactive arene oxide intermediate to form on the 2-substituted benzothiophene moiety. Substitution of the benzothiophene with other functional groups eliminated these potentially toxic metabolites. The data presented here demonstrate the utility of performing metabolic route screens early in the drug discovery process prior to lengthy and costly radiolabeled studies, and furthermore, implicate a 2-substituted benzothiophene moiety as a substrate for formation of a reactive arene oxide intermediate.