The binding ability of human serum albumin (HSA) on active pharmaceutical ingredients (APIs) is one of the most important parameters in the early stages of drug discovery. In this study, an immobilized HSA-based tool was developed for the rapid and easy in vitro screening of API binding. The work explored the serious incompleteness in the identification of HSA used for in vitro screening published in the last five years. To mitigate this problem, a comprehensive analysis and immobilization studies were performed on the most used HSA types. Serious differences in the colloidal stability of HSAs and their API binding ability on a selected set of APIs were observed. HSAs were immobilized on magnetic nanoparticles with glutardialdehyde (GDA) or cyclohexyl-diglycidyl ether (CDGE) linkers, which have never been used for HSA immobilization before. The HSA-MNP-CDGE complexes achieved a higher immobilization yield and preserved API binding ability
however, the esterase-like enzymatic activity of HSA reduced significantly.