In this paper, a new method for exergy analysis of distillation column is presented. It is based on the principle that there are exergy losses wherever driving forces exist. An approach has been developed for investigation the effects of various parameters on exergy losses. For verification of results, some experiments were performed using a pilot plant. The thermodynamic simulation of this pilot plant has been performed using a simulator. A thermodynamic model has been used to calculate the exergy losses of each stage in this simulation. The experimental and model results were observed to be accurate and in good agreement. The results showed that the use of the model results in more accurate analysis than the prevalent streamwise method exergy analysis in distillation column does. Both calculations and experiments showed that heat transfer in reboiler and condenser, and mass transfer are the two most significant sources of exergy losses.