Glaucoma is a chronic and progressive optic neuropathy characterised by damage of retinal ganglion cells. Evaluation of structural damage of the optic nerve is important in glaucoma diagnosis. The ISNT rule states that the neuroretinal rim width is generally widest in the inferior (I) area, followed by the superior (S) and nasal (N) areas, narrowest in the temporal (T) area. Myopia is a major risk factor for glaucoma. This study determined the applicability of inferior > superior > nasal > temporal (ISNT) rules on retinal nerve fibre layer (RNFL) thickness and rim area and evaluated the impact of various ocular factors on the performance of the ISNT rules in healthy myopic eyes. A total of 138 eyes from 138 healthy myopic subjects were included in this cross-sectional observational study. The peripapillary RNFL and optic disc in each eye were imaged with Cirrus HD optical coherence tomography (OCT) and Heidelberg Retina Tomograph II (HRT2), respectively. The performance of the inferior > superior (IS), inferior > superior > nasal > temporal (IST) and ISNT rules on RNFL thickness and rim area was determined and compared between low-to-moderate myopia and high myopia. The effects of ocular factors [including axial length, disc area, disc tilt, disc torsion, disc-fovea angle (DFA) and retina artery angle] on the performance of ISNT rules were evaluated with logistic regression analysis. The mean axial length and refractive error were 25.57±1.09 mm (range, 22.52–28.77 mm) and −5.12±2.30 D [range, −9.63 to −0.50 dioptres (D)], respectively. Sixty-three percent of the healthy eyes were compliant with the ISNT rule on rim area, while ISNT rule on RNFL thickness was followed in only 11.6% of the included eyes. They concluded that in healthy myopic subjects, 88.4% and 37% of eyes did not comply with the ISNT rule on RNFL thickness and rim area, respectively. Due to significant low compliance in healthy eyes, the ISNT rule and its variants have limited potential utility in diagnosing glaucoma in myopic subjects.