Fluorescent peptides combine the polypeptides with fluorescent groups organically to facilitate the measurement of enzyme activity. Here, we present a protocol to evaluate Sirtuin 7 (SIRT7) enzymatic activity by analyzing changes in luminescent signals from its substrate polypeptides. We outline the steps for SIRT7 protein purification, enzymatic reaction setup, fluorescence spectrum detection, and data analysis. Additionally, we describe two applications targeting SIRT7: high-throughput screening of SIRT7 inhibitors using microplate and the validation of potential SIRT7 inhibitors. For complete details on the use and execution of this protocol, please refer to Kang et al.(1).