AIM: To evaluate the reliability of β-III-Tubulin protein as a retinal ganglion cell (RGC) marker in the experimental glaucoma model. METHODS: Glaucoma mouse models were established by injecting polystyrene microbeads into the anterior chamber of C57BL/6J mice, then their retinas were obtained 14d and 28d after the intraocular pressure (IOP) was elevated. Retinal flat mounts and sections were double-labeled by fluorogold (FG) and β-III-Tubulin antibody or single-labeled by β-III-Tubulin antibody, then RGCs were counted and compared respectively. RESULTS: IOP of the injected eyes were elevated significantly and reached the peak at 22.8±0.7 mm Hg by day 14 after injection, then dropped to 11.3±0.7 mm Hg by day 28. RGC numbers counted by FG labeling and β-III-Tubulin antibody labeling were 64 807±4930 and 64614±5054 respectively in the control group, with no significant difference. By day 14, RGCs in the experimental group decreased significantly compared to the control group, but there was no significant difference between the FG labeling counting and the β-III-Tubulin antibody labeling counting either in the experimental group or in the control group. The result was similar by day 28, with further RGC loss. CONCLUSION: Our result suggested that the β-III-Tubulin protein was not affected by IOP elevation and can be used as a reliable marker for RGC in experimental models of glaucoma.