Researchers have paid much attention to small-scale natural fibers among the biological materials to seek innovative methods in order to create new high performance materials. Recently, spider dragline silk fibers are being studied because of their unique combination of high strength to weight ratio and high extensibility, which leads to a tough and lightweight fiber. Biomimetic fibers based on spider silk have been a focus of research for the past decade. However, there are still many unanswered questions about the mechanisms by which silk achieves its unique mechanical properties, as well as challenges in mechanical testing of electrospinning silk nanofibers which are often hindered by both small diameters and limited material availability. A method to characterize local mechanical behavior in small diameter nanofibers was developed to both improve understanding of structure-property in natural fibers and provide a method for comparing mechanical behavior in natural and electrospinning fibers.