Patients with beta-thalassemia major (BTM) suffer from fatigue, poor physical fitness, muscle weakness, lethargy, and cardiac complications which are related to an energy crisis. Carnitine and acylcarnitine derivatives play important roles in fatty acid oxidation, and deregulation of carnitine and acylcarnitine metabolism may lead to an energy crisis. The present study aimed to investigate carnitine and acylcarnitine metabolites to gain an insight into the pathophysiology of BTM. Dried blood spots of 45 patients with BTM and 96 age-matched healthy controls were extracted using a Neo-Mass AAAC kit (Labsystems Diagnostics Oy, Finland) and analyzed for free carnitine and 24 acylcarnitines by using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The findings indicated that the low level of free carnitine and acylcarnitines together with impaired CPT-1 activity contribute to energy crisis-related complications in patients with BTM.