The number of emerging novel stimulants modified based on beta-keto variations of amphetamine-like substances continues to rise. Dibutylone reports described in the medical and toxicological literature are limited, therefore little information is available in terms of quantitative confirmation or metabolism. During this study, authentic human specimens, including blood, urine, vitreous humor, oral fluid and liver were quantitatively and qualitatively analyzed for the presence of dibutylone and butylone, with paired case history and demographic information. Dibutylone concentrations were variable across all specimen types, specifically ranging from 10 to 1,400 ng/mL in postmortem blood specimens. The metabolic profile of dibutylone was mapped by in vitro incubation with human liver microsomes (HLM). Samples were analyzed using a SCIEX TripleTOF® 5600+ quadrupole time-of-flight mass spectrometer. Data processing was conducted using MetabolitePilot™. Authentic human specimens, including blood, urine, vitreous humor, oral fluid and liver, were utilized for in vivo verification of five HLM-generated metabolites in analytically confirmed cases of dibutylone use. Butylone was confirmed as a metabolite of dibutylone, but issues involving co-ingestion of these two novel stimulants or potential co-existence from synthesis lead to ineffectiveness as a true biomarker. Hydrogenation of the beta-ketone of dibutylone resulted in the most prominent metabolite found in human specimens, and its uniqueness to dibutylone over other stimulants leads to its classification as an appropriate biomarker for dibutylone ingestion. This is the first study to map the metabolic profile of dibutylone, including verification in authentic specimens, confirming metabolic conversion to butylone and identifying biomarkers more useful in forensic toxicological drug testing.