Human serum albumin (HSA) is used for mammalian embryo culture in assisted reproductive technology (ART) to adjust osmotic pressure and growth factor concentration in culture media. In human serum, transition metal ions, particularly Zn2+, bind to the coordination sites of HSA; thus analyzing the binding of these metal ions may help predict the possible harmful effects of these metals on embryo culture. In this study, we found that metal ions and zincon bound to HSA, and that a complex was formed with the HSA molecule. Using this method, the metals that bind to the multi-binding site of HSA, particularly Zn2+ can be evaluated by colorimetry in an ART laboratory. This analytical method is potentially applicable for metal detection in HSA in ART clinics and also for quick analysis of the coordination of 64Cu2+ with HSA as a probe chemical in positron emission tomography.