A measurement-based method to evaluate the efficiency of antennas used in microwave tissue imaging is proposed. The method is versatile and, in principle, applicable to antennas operating in free space as well. Two identical antennas are employed, one transmitting and the other one receiving. A signal flow graph (SFG) is developed for the two-port network formed by the antennas and the medium between them. The medium's electrical properties mimic those of tissue and can vary according to the application. Several measurements are performed with one of the antennas being loaded with known loads. A system of equations is constructed from the acquired data. The solution of this system produces the efficiency of the antenna in addition to other unknown parameters of the SFG. The efficiencies of two antennas are estimated from measured and simulated data and are verified through comparisons.