Lactoferrin is considered as a part of the innate immune system that plays a crucial role in preventing bac- terial growth, mostly via an iron sequestration mechanism. Recent data show that bovine lactoferrin prevents late-onset sepsis in preterm very low birth weight neonates by serving as an iron chelator for some bacterial strains; thus, it is very important to control the iron saturation level during diet supplementation. An accurate estimation of lactoferrin iron saturation is essential not only because of its clinical appli- cations but also for a wide range of biochemical experi- ments. A comprehensive method for the quantification of iron saturation in lactoferrin preparations was developed to obtain a calibration curve enabling the determination of iron saturation levels relying exclusively on the defined ratio of absorbances at 280 and 466 nm ( A 280/466 ). To achieve this goal, selected techniques such as spectrophotometry, ELISA,and ICP-MS were combined. The ability to obtain samples of lactoferrin with determination of its iron content in a simple and fast way has beenproven tobe veryuseful. Furthermore, a similar approach could easily be implemented to facilitate the determination of iron saturation level for other metalloproteins in which metal binding results in the appearance of a distinct band in the visible part of the spectrum.