Comparison of proteolytic activities of deconjugating enzymes toward ubiquitin or other ubiquitin-like modifiers (UbL), such as SUMO isoforms or Nedd8, has been a central focus in recent years to understand the dual or even multiple activities exhibited by members of the SENP/ULP family. This protocol outlines a competitive assay for an initial kinetic assessment of a SENP/ULP family member with specificity for alternative SUMO substrates. By using this method, the affinity of a SENP/ULP member for different SUMO isoforms or SUMO mutant variants can be compared. Also, IC50 values from competition experiments can be extracted to gain initial insights into the SENP/ULP substrate specificity. This is particularly useful when the KM(Michaelis constant) of the reaction is high and it is difficult to achieve high substrate concentrations. This protocol can also be applied to other CE-clan proteases with dual or multiple activities, such as those found in bacteria, to compare preferences between different types of UbL modifiers like ubiquitin, SUMO, or Nedd8.