INTRODUCTION: The diagnosis of Growth Hormone Deficiency (GHD) during childhood has been the subject of much controversy over the last few years. Aiming to accurate medical treatment, there is a need for biomarker discovery. OBJECTIVE: To characterize the metabolic profile of GHD children, examine the effect of GH administration on the metabolic signature, and investigate the correlations between metabolites and IGF-1. METHODS: Nuclear Magnetic Resonance (NMR)-based untargeted and targeted metabolomic approach applied to study the metabolic profiles of children with GHD. Plasma, serum, and urine samples were collected from twenty-two children diagnosed with GHD and forty-eight age matched controls from the Pediatric Endocrinology Unit of the University Hospital of Patras. Experimental data were examined by both multivariate and univariate statistical analysis. RESULTS: The results of this pilot study revealed a different metabolic fingerprint of children with GHD in comparison to age-matched healthy individuals. However, the detected alterations in the metabolite patterns before and after GH treatment were subtle and of minor discriminative statistical power. CONCLUSIONS: This study provides evidence that metabolome plays a pivotal role in GHD, but large-scale multicenter studies are warranted to validate the results.