The nano-indentation test was used to measure fracture toughness of the bone cement. The cement sample was prepared using two different mixing methods i.e., hand mixing and vacuum mixing. For this purpose, some cubic specimens, each of the size 10×10×5 mm3 were produced and then the nano-indentation test was performed on both the hand-mixed and the vacuum-mixed specimens by nano-indenter setup and atomic force microscopy observation. The fracture toughness values obtained from the hand-mixed and vacuum-mixed cements were compared. The results indicate that the vacuum-mixed cement has significantly higher fracture toughness compared with the hand-mixed ones. Since the nano-indentation test method needs less sample material, decreases costs and obtains reliable results, it can be considered as a suitable technique for determination of the mechanical properties of bone cements instead of the macroscale test methods.