Proteins are very important components in tears. Their phosphorylation is an important posttranslational modification affecting biological activity. Using proteomic techniques, this study was designed to analyze phosphoproteins found in open eye basal tears from normal human subjects. Proteins in tear samples were separated in 1-dimensional (1D) and 2-dimensional (2D) gels and phosphoproteins were selectively stained with Pro-Q diamond dye before visualization of all proteins using Sypro Ruby. Potential phosphoproteins in 2D gels were identified by liquid chromatography-mass spectrometry (LC-MS/MS) after trypsin digestion and phosphopeptide enrichment using titanium dioxide (TiO2) columns. The tryptic digests of the tear samples were also analyzed to identify phosphoproteins directly by LC-MS/MS after phosphopeptide enrichment. The major phosphoprotein stained by Pro-Q diamond in the gels and identified by LC-MS/MS from the spots was tear lipocalin. Tear lipocalin was separated into 3 different isoforms and one phosphorylation site (serine at position 24) was identified in one of the isoforms. Prolactin-induced protein, nucleobindin-2 and lipophilin C were also stained with Pro-Q diamond although no phosphorylated peptides from these proteins could be found using LC-MS/MS. Direct analysis of the tear tryptic digests by LC-MS/MS identified a further 12 potential phosphoproteins with tear lipocalin predominant. Four phosphorylation sites (position 24 (serine), 32 (serine), 34 (threonine) and 36 (tyrosine)) were identified for tear lipocalin using this method. These results indicate that tear lipocalin is the predominant phosphoprotein in normal human basal tears. Nucleobindin-2, prolactin-induced protein and lipophilin C also appear to be phosphorylated in basal tear samples.