The non-covalent interactions of gadolinium-based MRI contrast agents with macromolecules, such as human serum albumin (HSA), increase their efficacy. The identification of contrast agents that interact with HSA is a crucial first step in the complex, lengthy and expensive developmental process of a new potential HSA-targeting contrast agent. Fluorometry has been used as a possibly simpler and more effective tool of screening. In this study, the affinity of four compounds (Gd–DTPA, Gd–BOPTA, Gd–EOB–DTPA and MS-325) for HSA was investigated. The results show that the fluorescence method is a convenient tool that can easily detect this kind of non-covalent interaction owing to the small amount of required compound, the simplicity of the procedure and the popularity of the instrument, compared with the other approaches reported in the literature. However, fluorescence screening tests should be interpreted with caution since false-negative results will occur when the binding site of a gadolinium-based agent is far away from the location of the sole Trp residue of HSA or when an unsuitable site-marker is selected. Copyright © 2013 John Wiley & Sons, Ltd.