Polymer processing via electrospinning is a cost effective and scalable method for preparing nanofibers with industrial, electrical, and biomedical applications, particularly tissue engineering and drug delivery. Characterization methods for these fibers include microscopy techniques for vitro surface morphology information, spectroscopy methods to determine in vitro chemical composition, and medical imaging tools for in vivo assessment of morphology and efficacy of implanted material. The focus of this paper is be on recent applications for electrospun nanofibers, in vitro characterization methods, and medical imaging modalities that can be used for in vivo assessment of the fibers, as well as insights in how to adapt existing techniques toward the characterization of electrospun materials.