Enzyme characteristics, such as thermal stability and catalytic activity, can be improved in a targeted manner. However, the screening of target mutants is time-consuming and requires highly experimental downstream efforts. Here, we describe a simple strategy based on plasmid display and limited proteolysis for the rapid and easy screening of highly stable and active mutants from a library. When glutathione S-transferase was used as a model enzyme, the resulting mutants obtained in the first round of screening were approximately two- to sevenfold more thermostable than the wild-type enzyme at 50 A degrees C, with similar enzyme activity. This methodology is therefore powerful for the in vitro enrichment and screening of thermostable and active mutants. It can reduce downstream experimental effort and can create a high-quality library using relatively simple steps.