The mechanical properties of anterior skull base (ASB) bones are not well understood due to their complex geometry and deep location. However, it is of particular interest for skull base surgeons to appraise the force range they can apply during procedures and know what kind of haptic feedback a simulation device should produce in order to be realistic for trainees. The aim of this study was to establish a measurement protocol to set the level of hydration state, temperature and curve analysis method for spherical depth sensing nanoindentation of ASB bones. A definitive screening design method was used to test the different possible combinations of these factors. Two samples of ASB bones from the heads of two human body donors (two specimens) were selected according to their microstructure as assessed by micro-CT (microtomography):​ low-porosity (16.87%, sphenoid bone) and high-porosity (79.85%, ethmoid bone). Depth measurement series of 36 nanoindentations (n = 288) were performed on specimen 1 according to the L8 Taguchi orthogonal array to study the effect of temperature (two levels:​ 20 or 37 °C), hydration state (dry or immerged in physiological saline sodium chloride), and loading curve analysis according to the Hertzian contact theory (fitting at the start or at the end). The mean values of reduced Young's (E*) modulus varied significantly depending on the hydration status and bone microstructure. In order to obtain the physiological properties of ASB bones, we thus propose performing immersion tests. To simplify the experimentation protocol, future experiments must include a room temperature level and a fit of the curve at the end of the load. A validation series was performed on the second specimen to assess the set of parameters. The E* in dry bone gave mean values of 994.68 MPa, versus 409.79 MPa in immerged bones (p < 0.00001). This is the first time a study has been carried out on ASB bones, defining the experimental parameters related to physiological conditions.