The purpose of this study was to improve a mouse model of chronic intraocular pressure (IOP) elevation utilizing microbead injection in two strains of mice and to assess the effect of age and anesthesia on measured IOP. We compared our previous model with two modified protocols for injecting polystyrene microbeads and viscoelastic material in CD1or C57BL/6 mice. The measured outcomes were degree of IOP elevation and production of axonal loss. The first new protocol was injection of 3 μL of equal volumes of 6 μm and 1 μm diameter beads, followed by 2 μL of viscoelastic (3+2). The second new protocol injected 4 μL of the two bead mixture, then 1 μL of viscoelastic (4+1). Both were compared to injection of 2 μL of 6 μm beads with 3 μL of viscoelastic (2+3). We also compared the effects of age and of two anesthetic regimens (intraperitoneal ketamine/xylazine/acepromazine versus isoflurane gas) on measured IOP in untreated eyes of both strains. IOP was 2mm Hg lower with intraperitoneal than with gas anesthesia in both strains (p=0.003, p<0.0001, t-test). IOP measurements were lower in untreated young (2 months) compared to older (10 months) C57BL/6 mice (p=0.001, t-test). In the experimental glaucoma mouse model, mean IOP and number of elevated IOP measurements were higher in newer protocols. Mean axon loss with the 4+1 protocol (all strains) was twice that of the 2+3 and 3+2 protocols (36% vs. 15% loss, p=0.0026, ANOVA), and mean axon loss in CD1 mice (21%) was greater than in C57BL/6 mice (13%) (p=0.047, ANOVA). Median axon loss in 4+1 protocol treated C57BL/6 mice expressing yellow fluorescent protein in 2% of retinal ganglion cells (RGCs) had greater median axon loss than C57BL/6 4+1 protocol treated mice (26% vs. 10%, p=0.03). The 4+1 protocol provided higher, more consistent IOP elevation and greater axonal loss. The effects of age, strain, and anesthesia on induced IOP elevation and axon damage must be considered in mouse experimental glaucoma research.