We have developed a fast, reliable and easily reproducible semiautomated quantitative damage grading scheme to assess axonal loss in the optic nerve after inducing ocular hypertension using a laser glaucoma model in adult rats. This targeted sampling method has been validated against complete axon counts, and compares favorably with a conventional, random sampling, semiquantitative method. In addition, we present a standardized method to quantify axons in a semiautomated way, using freely available ImageJ software, and describe in detail the method used to induce glaucoma. Our techniques can be easily implemented in any laboratory, thanks to the public availability of the software and the simplicity of the method. Depending on the number of animals used in a particular study, the whole process from experimental elevation of intraocular pressure to tissue processing and data analysis should take ∼40 d.