Improved methods for non-invasive in vivo assessment are needed to guide development of animal models of atherosclerosis and to evaluate target engagement and in vivo efficacy of new drugs. Using novel 3D-micro-ultrasound technology, we developed and validated a novel protocol for 3D acquisition and analysis of imaging to follow lesion progression in atherosclerotic mice. The carotid arteries of ApoE receptor knockout mice and normal control mice were imaged within the proximal 2mm from the aortic branch point. Plaque volume along that length was quantified using a semi-automated 3D segmentation algorithm. Volumes derived by this method were compared to those calculated using 3-D histology post-mortem. Bland-Altman comparison revealed close correlation between these two measures of plaque volume. Furthermore, using a segmentation technique that captures early positive and 33 week negative remodeling, we found evidence that plaque volume increases linearly over time. Each animal and each plaque served as its own control, allowing accurate comparison. The high fidelity anatomical registration of this protocol provides increased spatial resolution and therefore greater sensitivity for measurement of plaque wall size, an advance over 2-dimensional measures of intimal-medial-thickening. Further, 3-dimensional analysis ensures a point of registration that captures functional markers in addition to the standard structural markers that characterize experimental atherosclerosis. In conclusion, this novel imaging protocol provides a non-invasive, accurate surrogate marker for experimental atherosclerosis over the life of the entire lesion.