This Application Note provides a comparative overview of real-time PCR reactions at 20 μl, 10 μl and 5 μl reaction volumes, each generated on the Mastercycler ep realplex real-time PCR system. SYBR® Green I- as well asTaqMan® probe-based assays were performed, and the reaction setup was done with the aid of the epMotion® 5070 automated pipetting system. It is shown that real-time PCR reactions with small reaction volumes can successfully be performed on the Mastercycler ep realplex: The reaction efficiencies obtained for the presented assays, including the lowest volume reactions, were comparably good and the Ct values obtained from the 10 μl- and 5 μl- reactions were only slightly higher than those detected for the 20 μl setups.