Designer benzodiazepines provide an attractive alternative to prescribed benzodiazepines for abuse purposes as they are readily available over the Internet at a low price. Although these compounds have been developed by pharmaceutical companies, at this time, they do not have a legal status. Their detection in biological fluids is challenging owing to the very few available data. For instance, there is no information about metizolam (also known as desmethyletizolam) analysis, distribution and metabolism in the scientific literature. Metizolam was ordered via Internet and a 2mg blue tablet was orally administered to a 54-year-old healthy man (85kg). Oral fluid (saliva) was collected over 8hours using the NeoSal™ device. Sweat was collected with the PharmChek™ sweat patch technology over 72hours. Exhaled breath was collected with the ExaBreath^® DrugTrap device over 15hours. Beard hair was collected 4 and 10 days after administration. Finally, head hair was collected 3 weeks after administration. Metizolam was detectable in saliva during 8hours, with concentrations lower than 1ng/mL (max at 0.77ng/mL after 2hours). Increasing concentrations up to 186pg/patch (after 72hours) of metizolam were detected in the sweat patches. Metizolam produced for 4hours a very low but significant chromatographic signal in exhaled breath, with concentrations always lower than 10pg/filter. Finally, metizolam tested positive in beard hair (0.73 and 0.28pg/mg after 4 and 10 days, respectively) and head hair (0.27pg/mg). From the data obtained from one subject, metizolam showed very low concentrations in various alternative matrices, attesting that the potential forensic implications (drug-facilitated crime, addiction, DUID…) of metizolam abuse must be considered as a new challenge for toxicologists.