Measuring fracture toughness for bone on the microscale is challenging due to the complex structure of bones which makes crack development unpredictable for traditional indentation methods. However, the development of a method to measure fracture toughness on a microscale can provide insights into how fracture toughness changes locally under different conditions, such as age or disease. In this study, we use the micropillar splitting method to determine the fracture toughness of bone from 24- week-old male mice femurs. Our findings examine heterogeneity within a whole bone, determine variance and repeatability and also compare microscale fracture toughness with whole bone fracture toughness. The findings reveal that the microscale fracture toughness does not vary with respect to the bone that formed at different ages. Additionally, microscale fracture toughness is lower than whole bone fracture toughness, most likely due to many extrinsic toughening mechanisms present in macroscale but not microscale. The micropillar splitting technique addresses some of the drawbacks of previously used notched three-point bending and double cantilever cracking methods, such as the need for SEM to measure the fracture length after the experiment and the influence of surface defects, such as pre-existing cracks or damage caused by the focused ion beam. The micropillar splitting approach provides a localized fracture toughness measurement with high precision and could be used to perform the fracture toughness measurement in hydrated conditions.