BACKGROUND. Due to the lack of understanding of the fascicular organisation, vagus nerve stimulation (VNS) leads to unwanted off-target effects. Micro-computed tomography (microCT) can be used to trace fascicles from periphery and image fascicular anatomy. NEW METHOD. In this study, we present a simple and reproducible method for imaging fascicles in peripheral nerves with iodine staining and microCT for the determination of fascicular anatomy and organisation. RESULTS. At the determined optimal pre-processing steps and scanning parameters, the microCT protocol allowed for segmentation and tracking of fascicles within the nerves. This was achieved after 24 hours and 120 hours of staining with Lugol’s solution (1% total iodine) for rat sciatic and pig vagus nerves, respectively, and the following scanning parameters - 4 μm voxel size, 35 kVp energy, 114 μA current, 4 W power, 0.25 fps in 4 s exposure time, 3176 projections and a molybdenum target. COMPARISON WITH EXISTING METHODS. This optimised method for imaging fascicles provides high-resolution, three-dimensional images and full imaging penetration depth not obtainable with methods typically used such as histology, magnetic resonance imaging and optical coherence tomography whilst obviating time-consuming pre-processing methods, the amount of memory required, destruction of the samples and the cost associated with current microCT methods. CONCLUSION. The optimised microCT protocol facilitates segmentation and tracking of the fascicles within the nerve. The resulting segmentation map of the functional anatomical organisation of the vagus nerve will enable selective VNS ultimately allowing for the avoidance of the off-target effects and improving its therapeutic efficacy.