Over the past decades, it has become abundantly clear that enzymes evolved to 
detoxify and eliminate foreign chemicals from the body, occasionally generate 
highly reactive metabolites which have toxicological implications. To decrease 
the probability of late clinical failure or market withdrawal, there has been an 
increased prioritization on understanding key metabolic processes that might 
cause drug interactions or toxicities. Significant advances have been made in 
the detection of reactive metabolites and in understanding the structure 
activity relationship. It is now widely accepted that compounds with certain 
functional groups such as anilines, quinones, hydrazines, thiophenes, furans, 
acylpropionic acids, and alkynes have a much greater associated risk towards 
formation of reactive metabolites than compounds that do not contain such 
"structural alerts". Detection of reactive metabolites is usually done with in 
vitro assays, which have become more sensitive with advances in mass 
spectrometry. As an increasingly large number of compounds that form reactive 
metabolites have been identified, much of the focus has shifted from detection 
to evaluation of toxicological implication. While there is a disproportionate 
number of compounds metabolized to reactive metabolites that are associated with 
drug-induced hepatotoxicity and serious skin toxicities such as toxic 
endothelial necrolysis and Steven's Johnson syndrome, attempts to predict 
toxicity based on in vitro testing have been discouraging. In this review we 
attempt to summarize the experimental options available to evaluate reactive 
metabolites.