Small-scale natural fibers are among the biological materials being studied by researchers seeking innovative methods to create new high performance materials. For example, 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 biomimetic silk fibers (which is often hindered by both small diameters and limited material availability). A method to characterize local mechanical behavior in small diameter fibers was developed to both improve understanding of structure-property relationships in natural fibers and provide a method for comparing mechanical behavior in natural and biomimetic fibers. The deformation mapping technique described in this paper, which utilizes a piezoelectric micromanipulator with pulled glass tips, an inverted microscope with attached camera, and an image processing MATLAB program, is also applicable to the characterization of other micro- and nanoscale fibers where local deformation mechanisms may be of interest (e.g., for mechanical characterization of electrospun fibers).