Among various cellular attributes modulating aqueous humor (AHAqueous humor (AH)) outflow through the trabecular pathway and eventually intraocular pressure, the involvement of actomyosin regulated cellular contraction and relaxation, cell-extracellular matrix adhesion and cell-cell junctions, and mechanotransduction are well recognized. Although various biological and pharmacological agent-modulated AHAqueous humor (AH) outflows were associated with altered actin cytoskeletal organization and cell adhesive interactions of trabecular meshwork (TM) cells, these changes were analyzed largely with a biased approach to examine the specific proteins, but there were very few efforts in examining them with hypothesis-free unbiased approach in their native state. Therefore, in this chapter, we describe a protocol tailored to characterize the cytoskeleton of TM cells. This simple protocol can be applied to identifying the differentially regulated TM cell cytoskeleton proteins under any given treatment condition in conjunction with quantitative proteomic analysis.