In this paper, a new image analysis based method for electrospun nanofiber diameter measurement has been presented. The method was tested by a simulated image with known characteristics and a real web. Mean (M) and standard deviation (STD) of fiber diameter obtained using this method for the simulated image were 15.02 and 4.80 pixels respectively, compared to the true values of 15.35 and 4.47 pixels. For the real web, applying the method resulted in M and STD of 324 and 50.4 nm which are extremely close to the values of 319 and 42 nm obtained using manual method. The results show that this approach is successful in making fast, accurate automated measurements of electrospun fiber diameters.