We employed a novel picoindenter (PI)/scanning electron microscopy (SEM) technique to measure the pull-off force of an individual electrospun poly(vinylidene fluoride) (PVDF) fibers. Individual fibers were deposited over a channel in a custom-designed silicon substrate, which was then attached to a picoindenter. The picoindenter was then positioned firmly on the sample stage of the SEM. The picoindenter tip laterally pushed individual fibers to measure the force required to detach it from the surface of substrate. SEM was used to visualize and document the process. The measured pull-off force ranged between 5.8 ± 0.2 μN to ~17.8 ± 0.2 μN for individual fibers with average diameter ranging from 0.8 to 2.3 μm. Thus, this study, a first of its kind, demonstrates the use of a picoindenter to measure the pull-off force of a single micro/nanofiber.