Purpose: To develop and evaluate an algorithm for computerized evaluation and measurement of the endometrial-myometrial junction (EMJ). Materials and Methods: The advanced image processing toolbox of the Matlab software package was used for identification and quantitative analysis of the EMJ area on three-dimensional (3D) rendered coronal plane uterine images, with clear-cut borders of the EMJ. The algorithm was used to process the images and calculate the geometric parameters characterizing the identified EMJ. The manual measurements of the maximum thickness of the EMJ were compared to automated measurements performed by the algorithm on the same images. Results: For all three interfaces, the mean maximum manual measurement was less than the mean maximum computed measurement. The differences between the two measurements were not statistically significant (p = 0.275, 0.608 and 0.419 for the right wall, left wall, and fundus, respectively). The mean systematic and random errors ranged from 5.4% to19.3% and 20.4% to 48.6%, respectively. Pearson correlations for the right wall, left wall and fundus (r = 0.642, p = 0.001; r = 0.730, p < 0.001, and r = 0.694, p < 0.001, respectively) were good. Conclusions: Maximum EMJ thickness measurements performed by the innovative Matlab software algorithm are as accurate as manual measurements, and have the potential to reduce inter-observer variability.