AH is a complex mixture of electrolytes, organic solutes, growth factors, cytokines, and proteins that provide the metabolic requirements to the avascular tissues of the anterior segment. ( , , )The protein component of AH is minimal ranging from 120 to 500 ng/?L. The proteins in AH arise from plasma as the result of filtration through fenestrated capillaries of the ciliary body stroma via the iris root. Although AH is a diffusate of plasma, but it has qualitative and quantitative differences in protein content in compared plasma. Identifying the protein component of tissues or fluids is vital to understanding the role these proteins have in normal physiology. AH proteins that are secreted from the anterior segment tissues may have a significant role in the pathogenesis of various eye diseases. ( , , , , )Characterization of the hAH proteome will provide new insights into the factors involved in maintaining anterior segment homeostasis and will also establish a foundation for biomarker discovery in various eye diseases of the anterior segment, such as glaucoma and corneal dystrophies. The Human Proteome Organization (HUPO) plasma proteome project database provided in the public domain by the University of Michigan Medical School contains more than 3000 proteins and protein isoforms. ( ) However, literature searches identified less than 150 proteins in hAH. Many of these proteins were identified as individual proteins based on targeted molecules of interest by Western blot analysis or enzyme-linked immunosorbent assay (ELISA). These were identified using proteomic approaches such as differential protein expression, Multidimensional Protein Identification Technology (MudPIT) ( ). Other studies using one- and two-dimensional gel electrophoresis coupled with mass spectrometry have relied mostly on comparative studies & differential protein expression ( , , ). However, these studies have confirmed only a few proteins across studies. Therefore, it is reasonable to suggest that little is known about the protein composition of hAH.Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS)is a powerful analytical tool to study peptides, proteins, and most other biomolecules (oligonucleotides, carbohydrates, natural products, and lipids) at femtomole levels and identify relevant biomarkers. ( , , )Sample preparation strategies are aimed at reducing sample complexity and improving quality of the measurements. A common method is solid-phase extraction (SPE) including affinity enrichment strategies to obtained peptide and protein fractions that can be mass analyzed by direct infusion into an electrospray ionization (ESI) source or by means of matrix-assisted laser desorption ionization (MALDI) without further need of time-consuming liquid chromatography (LC) separations. Functionalized magnetic beads are SPEs that due to their small size, offer the advantage of a higher concentration rate and therefore sensitivity due to their large binding surface area ( , ). Also the use of magnetic beads can be automated, providing a highly consistent high-throughput method . In this work, we aimed to search for differentially expressed proteins as potential biomarkers in primary congenital glaucoma patients using MALDI-TOF MS and SPE .