Many low-molecular weight proteins and peptides in human tears are potentially bioactive proteins but the range and number of these is yet to be fully characterized. A number of different sample preparation techniques were used to maximize the visualization of peptides from reflex tears. Samples were pretreated using precipitation and filtration techniques prior to analyses using MALDI-TOF MS. Peptides were searched for between 700 to 4000 m/z. Sample dilution in several different buffer systems followed by filtration with a 30-kDa cutoff filter and C18 reverse phase microcolumn purification produced significantly (p = 0.049) more peaks in tears than other methods used to prepare tears prior to MALDI-TOF MS. This study has established a technique for optimizing the visualization of naturally occurring peptides in tears.