The usefulness of the Inferior>Superior>Nasal>Temporal (ISNT) rule for the neuroretinal rim (NRR) has been widely used in differentiating normal eyes from glaucoma, but only a few studies have assessed whether this classical ISNT rule applies to the retinal nerve fibre layer (RNFL). This study aimed to determine the applicability of the ISNT rule for the peripapillary RNFL thickness in normal eyes using Spectral Domain Optical Coherence Tomography (SD-OCT) and assess if variants of the ISNT rule apply. A cross-sectional study was conducted on 120 eyes of 62 healthy subjects who fulfilled the study criteria. Peripapillary RNFL thickness was measured by OCT scan and each eye's global, superior, temporal, nasal, and inferior quadrant thickness was noted. The values obtained were analyzed to determine the percentage of eyes obeying the ISNT rule and its variants. The ISNT rule for RNFL thickness was applicable for normal subjects in only 53.33% of cases. Removing the nasal quadrant from analysis increased the number of eyes obeying the IST rule to 71.66%. Further exclusion of the temporal quadrant showed almost the same results (75%) for the IS rule. The ISNT rule for RNFL thickness could be validated in only 53.33% of normal individuals. Though documented as useful for NRR during ophthalmoscopy in glaucoma diagnosis, the ISNT rule did not apply to the quadrant values on RNFL on SD-OCT examination.