Elevated intraocular pressure (IOP) is the best recognized risk factor for the pathogenesis of glaucoma and the extent of retinal ganglion cell (RGC) degeneration in glaucoma is closely correlated with the extent of IOP elevation. Therefore, accurately and reliably measuring IOP is critical in investigating the mechanism of pressure-induced RGC damage in glaucoma. However, IOP is measured under general anesthesia in most studies using mouse models and many anesthetics affect the IOP measurements in both human and animals. In the present study, we used a noninvasive approach to measure the IOP of mice with normal and elevated IOP. The approach used mice that were awake and mice that were under general anesthesia. Our results demonstrate that not only the behavioral training enables IOP measurement from conscious mice without using a restrainer, it also significantly improves the consistency and reliability of the IOP measurement. In addition, we provide a direct comparison between awake and anesthetized IOP measurements as a function of time after the induction of general anesthesia with several commonly used anesthetic agents. We found that all tested general anesthetics significantly altered the IOP measurements both in normal eyes and in those with elevated IOP. Therefore, we conclude that behavioral training of mice can provide an approach to measure awake IOP that does not require general anesthesia and thus produces reliable and consistent results.