Background: Despite treatment with therapeutic hypothermia 45% of neonates with hypoxic-ischemic encephalopathy show an adverse outcome. Interventions combining hypothermia with additional neuroprotective strategies are needed in order to further improve the outcome of these neonates. The selective inhibitor of nNOS and iNOS 2-Iminobiotin has shown neuroprotective properties in animal models, but has not been combined with therapeutic hypothermia in humans. Methods: A single centre open label, prospective study will be performed. Term and near-term neonates treated with therapeutic hypothermia after perinatal asphyxia without major congenital malformations will be eligible for inclusion. In group A (N=6), patients will receive 4 bolus infusions of 2-iminobiotin (0.16 mg/kg) every 6 h. Short term safety will be assessed by monitoring of any changes in vital signs, aEEG or cerebral saturation using near-infrared spectrometry. For pharmacokinetic analyses, five blood samples of 0.4 ml per patient will be taken. Nonlinear mixed effects modelling (NONMEM) will be used to calculate individual area under the concentration time curves (AUC) as well as population parameters for clearance and distribution. After group A, an interim analysis will take place to determine the dosing regimen for group B (N=6). Discussion: This study will be the first to evaluate the use of 2-Iminobiotin in addition to therapeutic hypothermia in human neonates. Results from this study will contribute to the design and dosing regimen of a placebo controlled randomized controlled trial evaluating the efficacy of 2-Iminobiotin in this population as well as providing an understanding of 2-Iminobiotin pharmacokinetic parameters. Trail registration: European Clinical Trials Database (2014-004265-25) and the Netherlands Trial Register (www.trialregister.nl: NTR5221).