Transfusional iron overload associated with thalassemia leads to the appearance of non-transferrin-bound iron (NTBI) in blood that is toxic and causes morbidity and mortality via tissue damage. Hence, a highly sensitive and accurate assay of NTBI, with broad clinical application in both diagnosis and validation of treatment regimens for iron overload, is important. An assay based on iron chelation by a high-affinity siderophore, azotobactin, has been developed. The steps consist of blocking of native apotransferrin iron binding sites, mobilization of NTBI, ultrafiltration of all serum proteins, and finally the addition of the probe, which has a chromophore that fluoresces at 490 nm. Binding of Fe3+ to azotobactin quenches the fluorescence in a concentration-dependent manner. Measured NTBI levels in 63 sera ranged from 0.07 to 3.24 microM (0.375+/-0.028 microM [means+/-SEM]). It correlated well with serum iron and percentage transferrin saturation but not with serum ferritin. Pearson's correlation coefficients were found to be 0.6074 (P<0.0001) and 0.6102 (P<0.0001) for percentage transferrin saturation and total serum iron, respectively. The low values are due to the patients being under regular chelation therapy even prior to sampling, indicating that the method is sensitive to very low levels of NTBI, allowing a much lower detection limit than the available methods.