Quantification of bone strain can be used to better understand fracture risk, bone healing, and bone turnover. The objective of this work was to develop and validate an intensity matching image registration method to accurately measure and spatially resolve strain in vertebrae using imaging. A strain quantification method was developed that used two sequential scans, taken in loaded and unloaded configurations. The image correlation algorithm implemented was a multiresolution intensity matching deformable registration that found a series of affine mapping between the unloaded and loaded scans. Once the registration was completed, the displacement field and strain field were calculated from the mappings obtained. Validation was done in two distinct ways: the first was to look at how well the method could quantify zero strain; the second was to look at how the method was able to reproduce a known applied strain field. Analytically defined strain fields that linearly varied in space and strain fields resulting from finite element analysis were used to test the strain measurement algorithm. The deformable registration method showed very good agreement with all cases imposed, establishing a detection limit of 0.0004 strain and displaying agreement with the imposed strain cases (average ). The deformable registration routine developed was able to accurately measure both strain and displacement fields in whole rat vertebrae. A rigorous validation of any strain measurement method is needed that reports on the ability of the routine to measure strain in a variety of strain fields with differing spatial extents, within the structure of interest.