Nanofibers produced by electrospinning method are widely used for drug delivery, as tissue scaffolding materials and filtration purposes where specific pore characteristics are required. For continued growth in these areas, it is critical that the nanofibers be properly designed for these applications to prevent failure. Most of the current methods only provide an indirect way of determining pore structure parameters and contain inherent disadvantages. In this study, we developed a novel image analysis method for measuring pore characteristics of electrospun nanofiber webs. Five electrospun webs with different pore characteristics were analyzed by this method. The method is direct, very fast, and presents valuable and comprehensive information regarding pore structure parameters of the webs. Two sets of simulated images were generated to study the effects of web density, fiber diameter and its variations on pore characteristics. The results indicated that web density and fiber diameter significantly influence the pore characteristics, whereas the effect of fiber diameter variations was insignificant.