Recently, a major manufacturer recalled several lots of iron assay reagent due to positive bias of roughly 15%-30% and the cause remains unknown. This study investigated the root cause of this positive bias and evaluated a simple practical approach to improve the assay. Performance comparison of recalled and unimpacted iron assay kits was done utilizing calibrators, quality control (QC) materials, and 42 remnant patient samples. Spectral scan and trace elements analysis of R1 and R2 reagents was performed. Copper (Cu) and thiourea (TU) spiking experiments were utilized to elucidate the cause and prevention of positive bias seen with recalled lots. Iron measurements in QC materials and patient samples using recalled reagents generated a positive bias of 17.5% and 21%, respectively. Correspondingly, the recalled R2 reagents, but not R1, showed a rise in basal absorbance along with an unanticipated presence of Cu (22.7 µg/dL) and lead (7.5 µg/L). Cu spiking to recalled and unimpacted R2 reagent intensified the reagent color besides falsely increasing its absorbance, calibration factor, and patient iron measurements. Interestingly, addition of TU (65 mmol/L) to R2 reagent from unimpacted lot prevented the short-term and prolonged Cu-induced spurious rise in calibration factor and patient iron estimations. We conclude that accidental copper contamination of R2 reagent during manufacturing could be a reason underlying the positive bias in the recalled iron reagent lots. Addition of TU in ferene-containing R2 reagent is a simple and effective means to prevent Cu-induced false elevation in iron values.