β-Thalassemia is a widespread autosomal recessive blood disorder found in most parts of the world. Fetal hemoglobin (HbF), a form of hemoglobin is found in infants, replaced by adult hemoglobin (HbA) after birth. Hydroxyurea (HU) is one of the most effective HbF inducer used for the treatment of anemic diseases. We aimed to improve the understanding of HU therapy in β-thalassemia by metabonomics approach using 1H NMR spectroscopy. This study includes 40 cases of β-thalassemia before and after HU therapy along with 40 healthy as controls. Carr-Purcell-Meiboom-Gill (CPMG) sequence was used to identify forty-one putative metabolites. Generation of models like partial least square discriminant analysis (PLS-DA) and orthogonal projections to latent structures discriminant analysis (OPLS-DA) based on different metabolites including lipids, amino acids, glucose, fucose, isobutyrate, and glycerol revealed satisfactory outcomes with 85.2% and 91.1% classification rates, respectively. The concentration of these metabolites was altered in β-thalassemia samples. However, after HU treatment metabolic profile of same patients showed closeness towards healthy. Deviant metabolic pathways counting lipoprotein changes, glycolysis, TCA cycle, fatty acid and choline metabolisms were identified as having significant differences among study groups. Findings of this study may open a better way to monitor HU treatment effectiveness in β-thalassemia patients, as the results suggested that metabolic profile of β-thalassemia patients shows similarity towards normal profile after this therapy.