PURPOSE. To evaluate the impact of self-acting eyetracking and retest software on the reproducibility of retinal nerve fiber layer (RNFL) thickness measurements in glaucoma patients and healthy control subjects using Spectralis(®) SD-OCT. METHODS. RNFL thickness was measured in 56 normal and 47 glaucomatous eyes by one operator within one session with a brief rest between measurements. Three measurements were taken with eyetracker and retest function engaged (Method A), and three measurements were taken without eye tracker and without retest function (Method B). Measurements with Method A and Method B were taken alternately. RESULTS. Reliability, measured by intraclass correlation coefficient (ICC) for absolute agreement and coefficient of variation (COV), was calculated for the globalmean RNFL thickness (G), for each sector and for the peripapillaryboundle. The ICC (and lower 95% confidence interval (CI)) for the global mean RNFL thickness(G) for measurements using Method A in both normal and glaucomatous eyes was 0.99 (0.98 CI). In glaucomatous eyes the COV for measurements using Method B was between 2.7% and 10.5%, and between 1.3% and 3.5% formeasurements usingMethod A. CONCLUSION. Reproducibility of RNFL measurements with Spectralis(®) SD-OCT is excellent in bothnormal and glaucomatous eyes and can be significantly improved by using its eye trackerand retest software. The gain of reproducibility by using the software is significantly higher in glaucomatous eyes than in normal eyes. These findings suggest that software applications are capable ofsignificantly improving reproducibility of RNFL thickness measurements.