An instrument is provided to assess the transmurality of an ablation lesion from a first surface of a targeted biological tissue to an opposed second surface thereof. The instrument includes at least a first electrode operably attached to an ablation instrument adapted to engage a tissue first surface, and at least a second electrode adapted to engage a tissue second surface, the at least second electrode positioned generally opposed to the at least first electrode. Alternatively, the at least second electrode may be adapted to be placed within a chamber of an organ. These electrodes each measure at least one of conduction time, conduction velocity, phase angle, and impedance through at least a portion of the targeted tissue to determine the transmurality of the ablation lesion.