β thalassemia is the most prevalent autosomal recessive disorder characterized by absence or reduced production of hemoglobin (Hb) levels, primarily caused by mutations on β globin locus. β thalassemia is heterogeneous at the molecular level, presenting variable phenotypes accompanied with severe medical complications. Current standard of care for clinical management of β thalassemia includes regular, long-life safe blood transfusion along with appropriate iron chelation therapy. At present, the only permanent cure is bone marrow transplantation. An emerging and exciting therapeutic approach to handle β thalassaemia is production of fetal hemoglobin (HbF) which is major Hb of fetal life. In recent years, Hydroxyurea (HU) has proven to be a promising HbF augmenting agent but response to HU therapy varies from transfusion elimination to insignificant clinical response. Various approaches are being made to understand the mechanism HbF augmentation with differential responses. Advancement in proteomics offers an efficient tool to study differential proteome in response to treatment leading towards precision and personalized medicine. This study is designed to improve mechanistic understanding of proteomic changes that HU therapy exerted on β thalassemia patients, in consort with deciphering differential protein expression in HU responder and non-responder. Firstly, samples were subjected to twodimensional gel electrophoresis to assess differentially expressed proteins. Later, differential proteins were identified by label free quantitative proteomics approach. Two hundred and eighty seven proteins were identified with two or more unique peptides in samples studied. Among these, twenty eight proteins were found to be significantly different in pre versus post HU treated β thalassemia patients at probability of < 0.05. Eighteen proteins were down-regulated while ten were found to be up-regulated after HU treatment. Clinically important proteins include Hemopexin (HPX), Haptoglobin (HP), Haptoglobin-related protein (HPR), Hemoglobin subunit beta (HBB), Hemoglobin subunit delta (HBD), Hemoglobin subunit alpha (HBA1), Protein S100-A8 (S100A8), Apolipoprotein L1 (APOL1), Apolipoprotein C-I (APOC1), Transferrin receptor protein (TFRC), Complement C4-A (C4A), Apolipoprotein A (LPA), Ceruloplasmin (CP) and Ficolin-3 (FCN3). HU therapy in β thalassemia patients started reverting protein profile towards healthy pattern, in addition with decrease in transfusion requirements. A follow up study on plasma of HU treated β thalassemia patients was performed to compare proteomic profile of HU responder and non-responder. Twenty six proteins were found to be differentially expressed in HU responder versus non-responder at p < 0.05. Among these, fifteen proteins showed a significantly increased level while eleven proteins revealed a decreased in expression. Clinically relevant altered proteins in HU responder are Peroxiredoxin-2 (PRDX2), Carbonic anhydrase 1(CA1), Hemoglobin subunit gamma-1 (HBG1),Hemoglobin subunit beta (HBB), Hemoglobin subunit delta (HBD), Hemoglobin subunit alpha (HBA1), Properdin (CFP), Cholinesterase (BCHE), Phospholipid transfer protein (PLTP) and Plasma protease C1 inhibitor (SERPING1). We suggest that further research would be required for validation of identified proteins in large cohort to endorse as potential predictive biomarker for HU therapy. Considering the association of oxidative stress with β thalassemia, we also studied markers of oxidative stress in response to HU therapy in β thalassemia covering Paraoxonase1 (PON1), Reactive oxygen species (ROS), and Malondialdehyde (MDA). Although PON1 serve as an antioxidant to reduce the adverse effects of the oxidative stress in β thalassemia, our results indicate that mode of action of HU may not directly be through oxidative imbalance