Introduction: Advanced Normalization Tools (ANTs) is an image registration program that has been validated for analysis of several tissues, but not previously applied to the disc. The objectives of this study were to optimize ANTs for disc image registration and to validate ANTs strain measurements by comparison with Vic2D, a commercially available software previously applied to quantify disc strain, before and after nucleotomy. Methods: Human lumbar motion segments (n=5) underwent 1000 N compression before and after nucleotomy and MR images were acquired in a reference and deformed state. Image overlap statistics were used to select mapping parameters, and strain analysis was used to select the optimal number of splines. Once the optimal registration method was selected (elastic mapping, 0.01 outlier, 6x6 splines), axial and radial strains were measured in the AAF, PAF, and IVD. Results: Excellent overlap statistics were achieved. No significant differences were found for strains calculated with ANTs and Vic2D (p≥0.35). With nucleotomy, the axial compressive strain increased in the PAF (p=0.04) and there was a trend towards decrease in radial strain (p=0.07). Discussion: ANTs is an accurate and powerful tool to calculate disc strains from MR images.