The development of increasingly high-throughput and sensitive mass spectroscopy-based proteomic techniques provides new opportunities to examine the physiology and pathophysiology of many biologic fluids and tissues. The purpose of this study was to determine protein expression profiles of high-abundance synovial fluid (SF) proteins in health and in the prevalent joint disease osteoarthritis (OA). A cross-sectional study of 62 patients with early OA (n = 21), patients with late OA (n = 21), and control individuals (n = 20) was conducted. SF proteins were separated by using one-dimensional PAGE, and the in-gel digested proteins were analyzed by electrospray ionization tandem mass spectrometry. A total of 362 spots were examined and 135 high-abundance SF proteins were identified as being expressed across all three study cohorts. A total of 135 SF proteins were identified. Eighteen proteins were found to be significantly differentially expressed between control individuals and OA patients. Two subsets of OA that are not dependent on disease duration were identified using unsupervised analysis of the data. Several novel SF proteins were also identified. Our analyses demonstrate no disease duration-dependent differences in abundant protein composition of SF in OA, and we clearly identified two previously unappreciated yet distinct subsets of protein profiles in this disease cohort. Additionally, our findings reveal novel abundant protein species in healthy SF whose functional contribution to SF physiology was not previously recognized. Finally, our studies identify candidate biomarkers for OA with potential for use as highly sensitive and specific tests for diagnostic purposes or for evaluating therapeutic response.