The diagnosis of arginine vasopressin deficiency (AVD, formerly central diabetes insipidus) remains a challenge. In recent years, stimulated copeptin has emerged as a promising tool to diagnose AVD. In this single centre retrospective study, we identified paediatric patients with suspected pituitary insufficiency who underwent standard insulin tolerance testing (ITT) previously. Patients with AVD and non-matched controls without polyuria-polydipsia syndrome were identified. Diagnosis of AVD was confirmed retrospectively using comprehensive clinical and diagnostic characteristics. Serum copeptin concentrations were measured using a commercially available automated immunofluorescence assay (B.R.A.H.M.S Copeptin-proAVP KRYPTOR®) in -20°C stored samples collected before and 30, 45 and 60 minutes after insulin injection. Cut-off analyses were performed using ROC curves. 25 patients with AVD and 43 non-matched controls were available for analysis. Median basal copeptin concentrations of 1.51 pmol/l (IQR: 0.91-1.95; serum osmolarity: 288.5 mmol/l (IQR: 282.3-293.5) increased at a median of 30 minutes to a maximum of 1.95 pmol/l (IQR: 1.31-2.39) in AVD patients (p=0.113), and from 4.41 pmol/l (IQR: 3.36-6.68; serum osmolarity: 281.0 mmol/l (IQR: 274.0-286.0, p<0.001) to a maximum of 8.39 pmol/l (IQR: 4.95-19.72) (p<0.001) in controls. ROC analysis resulted in a cut-off of 3.0 pmol/l for maximum copeptin (91.7 % sensitivity, 94.1 % specificity) for the diagnosis of AVD. Our results suggest that insulin-stimulated serum copeptin concentrations are a sensitive and specific diagnostic tool for AVD in paediatric patients, which allows us to test multiple pituitary hormone axes simultaneously in a single test.