The coupling of separation by preparative ultracentrifugation and metal detection by inductively coupled plasma mass spectrometry (ICP-MS) has been explored for metal–protein equilibrium determinations. This study characterizes the stoichiometry as well as apparent (K app ) and intrinsic (K int ) binding affinities of the metal–protein association for a model protein. In particular, the affinity of Cu 2+ for the high affinity binding site in bovine serum albumin (BSA) is determined. Once equilibrium is established between Cu 2+ and BSA, preparative ultracentrifugation moves the metalloprotein away from the meniscus, leaving unbound equilibrium copper in the protein free solution. Since the initial (total) concentrations of purified BSA and Cu 2+ can be determined, the free copper concentration at equilibrium can also be determined by taking a small aliquot above the sedimenting boundary for analysis using ICP-MS. This analysis allows for the determination of free Cu 2+ ion, which is identical to the equilibrium concentration prior to ultracentrifugation. From these data K app and K int were determined at two different conditions, 100mM Tris(hydroxymethyl)aminomethane (Tris) at pH 9.53 and pH 7.93. log K app values of 17.6 and 14.6 were determined at pH 9.53 and pH 7.93, respectively. Furthermore, pH-independent log K int values of −1.43 and −1.04 were determined at pH 9.53 and 7.93, respectively. While the log K int at pH 9.53 was in good agreement with literature values obtained from alternative methods, K int at pH 7.93 was about 2.5× larger than previously reported. BSA undergoes a structural rearrangement between pH 7–9, and the generally accepted pH-dependency of protein tertiary structure may be responsible for the variations in the “intrinsic” binding constant. The Cu–BSA binding affinity was also monitored in 100mM Tris 0.1% sodium dodecyl sulfate (SDS) solution at pH 7.93 in order to determine the effect of a denaturant on metal binding. Results for both log K app and log K int were similar to those obtained in the absence of 0.1% SDS at pH 7.93. Overall, this study validates and shows the efficacy of combining preparative ultracentrifugation with ICP-MS detection for interrogating metal–protein associations while causing minimal equilibrium perturbations as a result of the separation and measurement processes. Advantages and disadvantages of this methodology are discussed as it relates to alternative methods for metal–protein studies.