Introduction: Iron overload in thalassaemia major patients mainly occurs due to periodic transfusions. When iron exceeds transferrin capacity, non-transferrin bound iron accumulates and causes tissue damage, including in the gastrointestinal tract, resulting in impaired enterocyte function and amino acid absorption. The aim of this study was to evaluate amino acid profiles in patients with thalassaemia major after repeated transfusions and chelation. Methods: Whole blood amino acids were analysed from dried blood spots using liquid chromatography tandem mass spectrometry. This study consisted of two parts: a cross-sectional and a cohort study in thalassaemia-β-major patients. In the cross-sectional study, amino acid profiles were analysed in 219 thalassaemia patients who received routine transfusion and chelation therapy, and 60 healthy control subjects. The cohort study included 21 subjects, from whom blood samples were taken at pre-transfusion, 1-day posttransfusion, one and three months post-chelation to evaluate changes in amino acid levels. Results: There were significant differences between amino acid levels in thalassaemia subjects and controls. Positive correlations were found between serum iron and transferrin with age, also between transferrin with proline, valine, phenylalanine, aspartic acid, and glutamic. Phenylalanine and aspartic acid were significantly lower in subjects with transferrin lower than 180 µg/dL. Significant correlations were found between haemoglobin with essential and non-essential amino acid groups. From the cohort study, significant changes were observed in glycine, alanine, leucine, and aspartic acid. Conclusion: Amino acid profiles in thalassaemia patients differed compared to healthy controls, even after transfusion and chelation. Phenylalanine and aspartic acid were significantly lower in subjects with low transferrin levels.