Background: Midazolam is a first-line drug for the treatment of status epilepticus, both by buccal and intravenous administration. In children and adolescents with obesity, midazolam pharmacokinetics may be altered, and the current dosing guidelines may therefore be insufficient. Objective: The objective of this study was to investigate the pharmacokinetics of midazolam, after intravenous administration, in obese and non-obese adolescents aged 11–18 years. Methods: All trial participants received a 1-µg midazolam microdose as an intravenous bolus. 13 blood samples were collected per participant at pre-specified timepoints. Plasma concentration–time data were fitted to pharmacokinetic models using non-linear mixed-effects modeling. Covariates such as weight, age, and body mass index standard deviation score were tested to explain the inter-individual variability associated with the pharmacokinetic parameters. Results: Sixty-seven adolescents were included in the analysis. The pharmacokinetics of midazolam was best described with a two-compartment model. The rate of distribution was faster, and the peripheral volume of distribution was larger in adolescents with a high body mass index standard deviation score compared with adolescents with a lower standard deviation score. Simulations revealed that long-term infusions based on total body weight could lead to high plasma concentrations in adolescents with obesity. Furthermore, simulated plasma concentrations after a fixed buccal dose indicated that adolescents with obesity may be at risk of sub-therapeutic midazolam plasma concentrations. Conclusions: The body mass index standard deviation score was shown to have a significant influence on the peripheral volume of distribution and the inter-compartmental clearance of midazolam. The current dosing guidelines for status epilepticus, where the midazolam dose is adjusted to total body weight or age, may lead to supra- and sub-therapeutic plasma concentrations, respectively, in adolescents with obesity. Trial registration: EudraCT: 2014-004554-34.