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Background: The prospects for children born with thalassemia currently include increased life expectancy and minimal or no organ damage. With the advances in the care of patients with β-thalassemia major (β-TM), there is a remarkable increasing prevalence of disrupted kidney functions due to various supposed factors. Therefore, early recognition of those likely to progress has become increasingly substantial. Serum creatinine and GFR, the current main diagnostic biomarkers, however, have several shortcomings for the evaluation of renal dysfunction. The aim of this study was to investigate novel urinary biomarkers including N-acetyl-b-Dglucosaminidase (NAG), neutrophil gelatinase-associated lipocalin (NGAL) urinary kidney injury molecule-1 (KIM-1), and liver-type fatty acid binding protein (L-FABP), to determine renal involvement in children with β-thalassemia major (β-TM). Methods: This prospective study involves measurements of the serum and urinary biochemical parameters, complete blood count, novel urinary biomarkers including urinary NAG to Creatinine (UNAG/Cr), UNGAL/Cr, UKIM-1/Cr and UL-FABP/Cr ratios and various demographic factors in 52 β-TM children and 29 healthy controls with comparable age and sex. Results: Patients had similar mean serum urea, creatinine and eGFR levels compared with controls (p>0.05 for all). The mean urinary protein to creatinine (UProtein/Cr) ratio was significantly higher in patients compared to the healthy subjects (0.13±0.09 mg/mg and 0.07±0.04 mg/mg, respectively; p=0.05). Significant correlations were found between urinary biomarkers and urinary electrolytes. Conclusion: Our results suggest that urinary NAG and NGAL may be