Auto-cascade refrigeration systems are the most used cycles to maintain spaces at ultra-low temperature conditions (-80 to −60 °C). The interest in these systems has risen due to the need to maintain and distribute COVID-19 vaccines. However, there is a lack of experimental validation of the theoretical models that are used to analyse and optimize them. In this work, a novel method to calculate the thermodynamic performance, circulating compositions, and energy efficiency of an auto-cascade refrigeration system is presented. The method is based on the measurements of an experimental auto-cascade plant at temperatures from −83.0 to −59.9 °C. The results of the study showed that the proposed calculation method was able to accurately predict the thermodynamic performance, circulating compositions and energy efficiency of an auto-cascade system. The uncertainty of the predictions was found to be below 1% for circulating compositions and 5.2% for the cooling capacity and COP.