Nanomaterials such as carbon nanotubes and graphene attract considerable attention due to their extraordinary mechanical and other properties. However, discontinuous nature of these carbon allotropes prevents easy transfer of their mechanical properties to the macro scale. Continuous nanofibers represent an emerging class of nanomaterials with critical advantages for structural and functional applications. However, their mechanical testing to date has been largely conducted using micrometer-long specimens in AFM-type or MEMS devices. In addition, most published reports did not test nanofibers through failure. As a result, information relevant to potential macroscopic structural applications of nanofibers is currently very limited. Here, we will present and discuss a recently developed, comprehensive mechanical evaluation protocol spanning controlled nanomanufacturing, handling, and mounting of long individual nanofiber specimens, as well as analysis of their large-deformation behavior through failure, and data reduction. The protocol will be demonstrated on several types of synthetic and biological nanofibers, including nanofibers exhibiting unique simultaneously ultrahigh elastic modulus, strength, and deformation to failure, resulting in superhigh toughness. The developed protocol will be instrumental for further optimization of mechanical properties of continuous nanofibers.