Purpose: To compare artifact rates in two-dimensional (2D) versus three-dimensional (3D) retinal nerve fiber layer (RNFL) scans using Spectralis optical coherence tomography (OCT) Methods: Thirteen artifact types in 2D and 3D RNFL scans were identified in 106 glaucomatous eyes and 95 normal eyes. Artifact rates were calculated per B-scan and per eye. In 3D volume scans, artifacts were counted only for the 97 B-scans used to calculate RNFL parameters for the 2.5–3.5-mm annulus. 3D RNFL measurements were calculated twice, once before and again after deletion of B-scans with artifacts and subsequent automated interpolation. Results: For 2D scans, artifacts were present in 58.5% of B-scans (62 of 106) in glaucomatous eyes. For 3D scans, a mean of 35.4% of B-scans (34.3 of 97 B-scans per volume scan) contained an artifact in 106 glaucomatous eyes. For 3D data of glaucoma patients, mean global RNFL thickness values were similar before and after interpolation (77.0 ± 11.6 μm vs. 75.1 ± 11.2 μm, respectively; P = 0.23). Fewer clinically significant artifacts were noted in 3D RNFL scans, where only 7.5% of glaucomatous eyes (8 of 106) and 0% of normal eyes (0 of 95) had artifacts, compared to 2D RNFL scans, where 58.5% of glaucomatous eyes (62 of 106) and 14.7% of normal eyes (14 of 95) had artifacts. Conclusions: Compared to 2D RNFL scans, 3D RNFL volume scans less often require manual correction to obtain accurate measurements. Translational Relevance: 3D RNFL volume scans have fewer clinically significant artifacts compared to 2D RNFL thickness scans.