Purpose: The purpose of this work was to examine the tear film proteome using a combination of one-dimensional (1D) and two dimensional (2D) gel electrophoresis and mass spectrometry-based techniques and to explore the effect of the tear collection methods on the tear proteome. Methods: Tear samples from eight normal non-contact lens wearing human subjects collected by Drummond glass microcapillary and Schirmer strips were subjected to 1D-sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), 2D-SDS-PAGE, and 2D LC-MS/MS (Multidimensional protein identification technology - MudPIT). Bands or cores from the 1D- and 2D-SDS-PAGE were cut digested with trypsin, and analyzed by tandem mass spectrometry for identification by the generation of sequence tags. Results: In total (across sampling and proteomic methods), 97 unique proteins were observed, and a significant number of the spots/bands in the PAGE were from posttranslational modifications. Fifty-four unique proteins were identified from proteins extracted from the Schirmer strips in comparison to 13 unique proteins identified from capillary tubes, and 30 unique proteins were identified by both collection methods. Secreted (serum) proteins were predominantly observed from tears collected by capillary whereas a combination of cellular and serum proteins were identified from tear film collected by Schirmer strips. Conclusions: Overall, these results suggest that the tear film collection and the proteomic method impacts the proteins present in the tear film and that care should be exercised in choosing a tear collection method to best correlate to the experiment being conducted or the hypothesis that is being tested. © 2008 Molecular Vision.