While nanoindentation is an ideal tool for mechanically testing at the “tissue-level” of bone, unique challenges exist in both testing methods and analytical approaches. Bone is heterogeneous in composition and structure. Bone’s hierarchical organization, typical of many biological tissues, implies that multiple structural levels may inﬂuence a single nanoindentation site. Mineral forms a complicated 3-D structure within and around a fibrillar collagen matrix. The degree of organization and mineral infilling varies with factors including the age, type, and location of bone material. Pores sized from nanometers to millimeters contain interstitial ﬂuid that interacts electrochemically and structurally with bone’s material constituents, adding further complexities to bone’s mechanical response. This chapter reviews practices for the collection of high quality data at small scales where the organization of bone’s three phases complicate our ability to prepare samples, visualize 3-D structure, and obtain exact measurements of material properties at small length scales. Nanoindentation, in combination with additional independent measures of the tissue, enable the detailed study of bone’s poorly understood 3-D construction, material properties, and mechanical behavior at sub-micrometer scales. This chapter addresses the use of nanoindentation to study bone through consideration of the interactions within and between the collagen, mineral and water phases.