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In situations where the molecular mechanism of many ocular disorders is unknown, owing to the difficulties associated with sampling from ocular tissues, human tear film can be a promising medium in ophthalmic research. The present study demonstrates an in-depth gel-based proteome optimization survey to approach more appropriate and efficient systems in various situations such as normal and dry-eye syndromes. Therefore, systematic and statistical evaluations were performed on different preparation methods, including acetone, acetone-methanol, chloroform-methanol-water, trichloroacetic acid (TCA)-acetone, tri-n-butylphosphate-acetone-methanol precipitations and ammonium sulfate fractionation at three different percentages of saturations (50, 70 and 90%). Methods were compared quantitatively on both one- and two-dimensional patterns. Some important parameters such as total protein recovery yield, densitometric analysis of some protein contaminants, banding patterns and total spot numbers along with statistical models for proper clustering were considered. Findings revealed noticeable impacts of preparation methods on all aspects of gel-based separations as well as recovery yield (ranging from 5.29 ± 0.96 to 22.56 ± 1.77 µg/mm) and banding and pattern resolution. In addition to all these, the most important point is that the total protein spot number on the final two-dimensional patterns (varied from 528.00 ± 19.00 to 657.00 ± 21.52 for different methods) were also noticeably increased in comparison with previously published reports (maximum of 250 spots), which is essential for a more comprehensive analysis. Increasing the proteome coverage in the present study is supposed to originate from improved solubility and effective rehydration during the sample application and isoelectric focusing (IEF) procedure along with proper sample preparation., (Copyright © 2014 John Wiley & Sons, Ltd.)