ß-thalassemia is the most common genetic disorder caused by mutations in β-globin gene resulting in absence/reduced production of Adult Hemoglobin (HbA). Regular blood transfusions and effective iron chelation is necessary for survival of β-thalassemia patients. An emerging therapeutic approach to handle β thalassaemia is production of fetal hemoglobin (HbF). Hydroxyurea (HU), a potent HbF inducing agent, has shown to be an effective drug for β-thalassemia management with variable response from transfusion independence to null outcome. Diversified response of β-thalassemia patients to HU therapy remains ambiguous for hematologist. Clinical proteomics has revolutionized the study of differential protein expression associated with disease and offers a unique technique to monitor the response to treatment. In current study, we focused on comparative analysis of plasma proteome in pre- and post- HU-treated β-thalassemia patients, as well as responders and non-responders to HU treatment. Plasma was collected from β-thalassemia patients before and after 6 months of HU treatment, and the treated group were sub-categorized on the basis of response to HU. Label-free LC-MS/MS analysis was performed on nano-HPLC coupled Q-Exactive Orbitrap Plus mass spectrometer. Quantitative data and statistical analysis was carried out using MaxQuant and Perseus respectively. Proteomics analysis revealed identification of 400 proteins in all groups; among them the expression of twenty eight proteins were significantly different in pre- vs post- HU treated groups, with, twenty six proteins being differentially expressed in responder vs non-responder groups. Transferrin receptor protein 1 (TfR) was the most down-regulated and hemopexin and haptoglobin were the most up-regulated proteins in plasma after HU treatment whereas proteins that differed significantly among responder and non-responder were carbonic anhydrase 1, hemoglobin subunit gamma-1 and peroxiredoxin-2.