Parathyroid hormone (amino acids 1-34) (PTH) regulates bone and calcium homeostasis. The magnitude of the effects of PTH on bone varies in osteoporosis patients. We employed ProteinChip technology to generate protein profiles from sera of mice treated once daily with PTH or vehicle for 3 or 11 days. Data analyses on selected arrays indicated significant increases in serum proteins or peptides in PTH-treated groups, compared to vehicle-controls. The magnitude of change increased with duration of treatment. Anion-exchange fractionation of sera prior to profiling on array surfaces increased the number of proteins detected that were regulated by PTH. The optimized purification conditions developed "on-chip" for subsets of proteins, reflected corresponding behavior with process-compatible chromatographic resins under elution chromatography. We have identified and evaluated subsets of serum proteins regulated by PTH treatment, using a combination of ProteinChip technology, column chromatography, PAGE and LC-MS/MS. Our data demonstrate the feasibility of using a panel of serum proteins to detect PTH responsiveness in humans.