Fuel cell is an efficient energy conversion device converting chemical energy directly into electrical energy. It is a fact that, to boost the fuel cell performance, resistance (charge transfer resistance, mass transfer resistance and electrolyte resistance) should be decreased. For this, many techniques have been used for cell testing such as: cyclic voltammetry, current interruption measurement, chronoamperometry, chronopotentiometry, polarization curve and electrochemical impedance spectroscopy (EIS). Among these techniques, EIS is a well-established, non-intrusive, non-destructive, semi-quantitative, and an efficient technique for identification of each circuit element. In this review article, application of electrochemical impedance spectroscopy in identification of individual components of total resistance and their dependence on different factors in biofuel cell along with some recent advancement in this technique have been discussed. Copyright (C) 2014, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.