To accelerate the metabolite identification process, there is an ongoing search for fully MS-based techniques that provide structural information on MS-detected ions. Among these technologies, infrared ion spectroscopy (IRIS) is a promising candidate, as it provides IR spectra for massisolated m/z features. Vibrational resonances are highly sensitive to details of the molecular structure, such that closely related isomeric molecular ions can often be differentiated on the basis of their IR spectra. Indeed, a range of studies have shown that IRIS can distinguish isomeric small molecules , including several phase-I drug metabolites . A significant advantage in comparison to techniques based on MS/MS fragmentation patterns is that gas-phase IR spectra can be reliably and efficiently predicted using quantum-chemical calculations based on density functional theory (DFT). Therefore, molecular structures can often be elucidated in a reference-free manner; for final confirmation or quantification purposes, only a single synthetic standard is then required. Previous work has demonstrated IRIS-based differentiation of ortho-, meta-and para-isomers that may result from the hydroxylation of drug compounds containing a phenyl moiety. In one of these studies, metabolites were extracted and analyzed from an in vitro drug incubation sample using a combination of LC-MS and IRIS. However, a drug molecule often undergoes several phase I (oxidation, hydrolysis or reduction) and phase II (conjugation) biotransformation reactions that occur sequentially. Elucidating a metabolic pathway involves identification of each of the (intermediate) metabolites and their place in the metabolic pathway. To explore the potential of IRIS for the mapping of full metabolic pathways, we demonstrate here the identification of several metabolites resulting from the incubation of midazolam in human hepatocytes. Midazolam (MDZ) is a widely used benzodiazepine and is known to metabolize via hydroxylation and glucuronidation reactions. Here, we detect several MDZ metabolites using LC-MS and elucidate their full molecular structures in absence of reference standards, using a combination of MS/MS fragmentation patterns and comparison of experimental IR spectra to DFT computed reference spectra. Glucuronated drug molecules readily expel the glucuronide-group upon collisional activation in an MS/MS experiment, challenging the identification of the site of glucuronidation. Here, we use this feature of glucuronides by recording IR spectra for both the glucuronated precursor ion and its glucuronide-loss MS/MS fragment (aglycone). This allows us to suggest a metabolic link between phase I and phase II metabolites and to narrow down the list of candidate structures for the glucuronide metabolite. Subsequently, the location of the glucuronide group can be determined from the precursor ion IR spectrum. MDZ is well-known to undergo a ring-opening in an acidic environment. As a further demonstration of the potential of IRIS for the identification of drug derivatives, we identify several ring-opened MDZ derivatives.