The manual methods are often used for measurement of fiber diameter of electrospun membranes, however these methods are not suitable for online quality control. Although number of image analysis methods have been reported for automated measurement of fiber diameter, but there is no literature available regarding reliability, accuracy and simplicity of these measurements. In this study, the distance transform methods suggested by Pourdeyhimi and Ziabari, direct tracking method, DiameterJ software and Zhang method were compared for automated measurement of fiber diameter of electrospun nanofibrous membranes. From one way ANOVA, the difference between the results of fiber diameter was found highly significant. The mean and standard deviation of fiber diameter obtained in case of Ziabari method were extremely close to the results of manual method and also to the true values of the simulated image. Therefore, the more accurate estimation of fiber diameter was confirmed in case of Ziabari method.