Presented herein, in certain embodiments, are approaches for robust bone splitting and segmentation in the context of small animal imaging, for example, microCT imaging. In certain embodiments, a method for calculating and applying single and hybrid second-derivative splitting filters to gray-scale images and binary bone masks is described. These filters can accurately identify the split lines/planes of the bones even for low-resolution data, and hence accurately morphologically disconnect the individual bones. The split bones can then be used as seeds in region growing techniques such as marker-controlled watershed segmentation. With this approach, the bones can be segmented with much higher robustness and accuracy compared to prior art methods.