Aims: Altered physiology caused by critical illness may change midazolam pharmacokinetics (PK) and thereby result in adverse reactions and outcomes in this vulnerable patient population. This study set out to determine which critical illness related factors impact on midazolam pharmacokinetics in children using population PK modelling. Methods: An observational, prospective, controlled study of children admitted to University Hospitals Leicester NHS Trust receiving IV midazolam as part of routine care. Children recruited into the study were either critically-ill receiving continuous infusions of midazolam or otherwise well, admitted for elective day-case surgery (control) who received a single IV bolus dose of midazolam. Blood midazolam and 1-OH-midazolam concentration and patient clinical data were used to develop a population PK model and to determine covariates which affect midazolam disposition during critical illness. Results: 35 patients were recruited into the critically ill arm of the study, 54 children into the control arm. Modelling demonstrated a significant change in midazolam clearance with acute inflammation (measured using C-Reactive Protein, CRP), cardio-vascular status and weight. Simulations predict that elevated CRP and compromised cardiovascular function in critically ill children result in midazolam concentrations up to 10-fold higher than healthy children. Conclusions: Raised CRP and compromised cardiovascular function significantly reduce midazolam clearance. The extremely high levels of midazolam observed in some critically-ill children indicate that the current therapeutic dosing regimen for midazolam can lead to over-dosing. Clinicians should be aware of this risk and intensify monitoring of such patients.