β-Thalassemia is one of the most prevalent forms of congenital blood disorders characterized by reduced hemoglobin levels with severe complications, affecting all dimensions of life. In addition, the mechanisms underlying the phenotypic heterogeneity of β-thalassemia are still poorly understood. Current standard treatment of β-thalassemia (i.e., blood transfusion, iron chelation, splenectomy, and treatment of complications) is based on symptoms of each patient. Augmentation of fetal hemoglobin (HbF) production has been an enduring therapeutic objective in β-thalassemia patients for which hydroxyurea (HU) have largely been the drug of choice and the most cost-effective approach. Advancements in metabolomics offer an efficient solution to study the complexity of diseases at the molecular level that expands treatment strategies. This study is designed on metabolite profiling to improve mechanistic understanding of phenotypic heterogeneity of β-thalassemia and hence better management of this disorder. Firstly, untargeted serum metabolites were analyzed after protein precipitation and SPE (solid phase extraction) using gas chromatography-mass spectrometry (GC-MS). 40 metabolites were identified having a significant difference between patients and control at probability of 0.05 and fold change >1.5. 17 were up-regulated while 23 were down-regulated. PCA and PLS-DA models revealed a fine separation with a sensitivity of 70% and specificity of 100% on external validation of samples. Metabolic pathway analysis revealed alteration in multiple pathways. Then, a follow up metabolomics study on the serum of 40 β-thalassemia patients before and after HU administration was done along with 70 other HU treated samples and healthy controls. The HU treated patients were further classified into good (GR), partial (PR) and non-responders (NR) according to their response. 25 metabolites that were altered before HU therapy at p ≤0.05 and fold change >2.0 in β-thalassemia patients; started reverting towards healthy after HU treatment. Metabolic pathway analysis also revealed that metabolism of linoleic acid, glycerolipid, glycerophospholipid, galactose, fatty acid biosynthesis, metabolism and their elongation in mitochondria were also reverted after HU therapy. As fatty acids (FAs) are not only involved in maintaining membrane integrity but also have biological activities that influence cell and tissue metabolism, function, and responsiveness to hormonal and other signals. Therefore, finally targeted quantification of thirteen free fatty acids was carried out to disclose the prognosis of HU in β-thalassemia using the combination of random forest (RF) with gas chromatography-multiple reaction monitoring-mass spectrometry (GC-MRM-MS). Docosanoic acid (C22:0) was most significantly altered in β-thalassemia as compared to healthy while erucic acid (C22:1 Δcis-13) can be used as potential marker of HU prognosis because its level became significantly dissimilar in same patients in response to HU. However, all three groups i.e. healthy, β-thalassemia and β-thalassemia after HU treatment differ because of nervonic acid (C24:1 Δcis-15). Moreover, HU therapy also increased apo A1 and decreased apo B protein levels. In inference, we noticed that HU therapy not only reduces need of blood transfusion in β-thalassemia but also rectifies the serum fatty acid profile and lowered the apolipoproteins ratio (apo B/apo A1), thus reducing the risk of CVD in these patients.