AbstractThe identification of biotransformation products of drug compounds is a crucial step in drug development. Over the last decades, liquid chromatography‐mass spectrometry (LC‐MS) has become the method of choice for metabolite profiling because of its high sensitivity and selectivity. However, determining the full molecular structure of the detected metabolites, including the exact biotransformation site, remains challenging on the basis of MS alone. Here we explore infrared ion spectroscopy (IRIS) as a novel MS‐based method for the elucidation of metabolic pathways in drug metabolism research. Using the drug midazolam as an example, we identify several biotransformation products directly from an in vitro drug incubation sample. We show that IR spectra of the aglycone MS/MS fragment ions of glucuronide metabolites establish a direct link between detected phase I and phase II metabolites. Moreover, using quantum‐chemically computed IR spectra of candidate structures, we are able to assign the exact sites of biotransformation in absence of reference standards. Additionally, we demonstrate the utility of IRIS for structural elucidation by identifying several ring‐opened midazolam derivatives formed in an acidic environment.