The new Beckman Coulter Optima AUC instrument, which features multi-wavelength detection that couples the hydrodynamic separation of colloidal mixtures to spectral deconvolution of interacting and non-interacting solutes present in a mixture, was used to analyze the composition of human serum albumin (HSA) bound to metallo-protoporphyrin. We present new methods implemented in UltraScan that permit Optima AUC-derived multi-wavelength data to be spectrally decomposed in the same fashion as has been made possible for the Cölfen detector earlier. We demonstrate this approach by spectrally separating sedimentation velocity experimental data from mixtures of apo-HSA and HSA complexed to different metallo-protoporphyrins. We further demonstrate how multi-wavelength AUC can accurately recover percentages of metallo-protoporphyrin-bound HSA and apo-HSA from mixtures and how multi-wavelength AUC permits the calculation of molar extinction coefficients for porphyrins bound to HSA. The presented method has broad applicability to other complex systems where mixtures of molecules with different spectral properties need to be characterized.