No means exist to evaluate the activity status, turnover, and prognosis of idiopathic osteonecrosis of the femoral head (IONFH) except for X-ray evidence of segmental collapse as a very good marker for prognosis. Moreover, the only current method for diagnosis of this disease is through physical examination and diagnostic imaging results, and no serum biochemical markers exist. A comparative analysis of serum proteomes was performed to discover proteins associated with osteonecrosis of the femoral head. Two-dimensional electrophoresis (2-DE) patterns of human sera from 10 patients with IONFH and 10 normal subjects were analyzed. The differentially expressed spots were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and 7 proteins were found. The expression levels of tissue-type plasminogen activator (t-PA), bone-carboxyglutamate protein (BGP), c-sis, and an unknown protein were downregulated in the sera of patients with IONFH, whereas the other 3 proteins, including plasminogen activator inhibitor type 1 (PAI-1), crosslaps, and anti-p53 antibody, were upregulated. To examine their applicability as diagnostic markers, levels of the 6 identified proteins in serum were validated from patients with IONFH, osteoarthritis (OA), rheumatoid arthritis (RA), and fracture using the enzyme-linked immunosorbent assay (ELISA) method. It was found that only serum levels of t-PA, PAI-1, crosslaps, and anti-p53 antibody in patients with IONFH were always significantly different from those in patients with OA, RA, and fracture. These results suggest that serum levels of t-PA, PAI-1, crosslaps, and anti-p53 antibody could be used as noninvasive diagnostic biomarkers for IONFH.