Ocular hypertension is a serious adverse effect of extended exposure to steroids, and may predispose a person to steroid induced glaucoma and permanent vision loss. Dexamethasone, a common glucocorticoid used to treat ocular inflammation, is known to induce ocular hypertension at the level of the trabecular meshwork (TM) and lead to steroid induced glaucoma via retinal ganglion cell death. Previous studies on dexamethasone treated TM cells reported increased fibrotic markers including alpha smooth muscle actin, fibronectin, and myocilin. However, there are no definitive studies showing a contractile phenotype of TM cells, another marker of fibrosis. To address this question, we adapted and implemented a 3D collagen hydrogen contractility assay to assess the contractile state of dexamethasone treated primary human trabecular meshwork (HTM) cells. Dexamethasone treated collagen hydrogel beads containing HTM cells had an average decrease of 35% in area compared to an 11% decrease in vehicle treated beads. This study may add further information on the mechanical changes dexamethasone induces on TM cells, and how it may contribute to the pathophysiology of steroid induced glaucoma.