Background. Software guided optic nerve assessment can assist in process automation and reduce interobserver disagreement. We tested depth analysis software (DAS) in assessing optic nerve cup-to-disc ratio (VCD) from stereoscopic optic nerve images (SONI) of normal eyes. Methods. In a prospective study, simultaneous SONI from normal subjects were collected during telemedicine screenings using a Kowa 3Wx nonmydriatic simultaneous stereoscopic retinal camera (Tokyo, Japan). VCD was determined from SONI pairs and proprietary pixel DAS (Kowa Inc., Tokyo, Japan) after disc and cup contour line placement. A nonstereoscopic VCD was determined using the right channel of a stereo pair. Mean, standard deviation, t-test, and the intraclass correlation coefficient (ICCC) were calculated. Results. 32 patients had mean age of 40±14 years. Mean VCD on SONI was 0.36±0.09, with DAS 0.38±0.08, and with nonstereoscopic 0.29±0.12. The difference between stereoscopic and DAS assisted was not significant (p=0.45). ICCC showed agreement between stereoscopic and software VCD assessment. Mean VCD difference was significant between nonstereoscopic and stereoscopic (p<0.05) and nonstereoscopic and DAS (p<0.005) recordings. Conclusions. DAS successfully assessed SONI and showed a high degree of correlation to physician-determined stereoscopic VCD.