High-throughput X-ray absorption spectroscopy was used to measure transition metal content based on quantitative detection of X-ray fluorescence signals for 3879 purified proteins from several hundred different protein families generated by the New York SGX Research Center for Structural Genomics. Approximately 9% of the proteins analyzed showed the presence of transition metal atoms (Zn, Cu, Ni, Co, Fe, or Mn) in stoichiometric amounts. The method is highly automated and highly reliable based on comparison of the results to crystal structure data derived from the same protein set. To leverage the experimental metalloprotein annotations, we used a sequence-based de novo prediction method, MetalDetector, to identify Cys and His residues that bind to transition metals for the redundancy reduced subset of 2411 sequences sharing <70% sequence identity and having at least one His or Cys. As the HT-XAS identifies metal type and protein binding, while the bioinformatics analysis identifies metal- binding residues, the results were combined to identify putative metal-binding sites in the proteins and their associated families. We explored the combination of this data with homology models to generate detailed structure models of metal-binding sites for representative proteins. Finally, we used extended X-ray absorption fine structure data from two of the purified Zn metalloproteins to validate predicted metalloprotein binding site structures. This combination of experimental and bioinformatics approaches provides comprehensive active site analysis on the genome scale for metalloproteins as a class, revealing new insights into metalloprotein structure and function.