Nanomaterial electrocatalysis is a critical field for advancing sustainable energy technologies such as carbon dioxide reduction and fuel cells, yet determining the active species is challenging because catalysts often evolve dynamically during a reaction. In this work, we investigate reductive deligation, a well-known activation process of ligated nanomaterials, on [Au25(PET)18]- (PET = 2-phenylethanethiol) nanoclusters under varying electrochemical conditions. We introduce a novel application of electrochemical impedance spectroscopy (EIS) to characterize the gold nanoclusters (AuNC) in situ throughout the reductive deligation process, which we term in operando EIS. This approach enables real-time monitoring of ligand removal by extracting key parameters such as the charge-transfer resistance. By systematically varying applied potential and pH, we gain kinetic and mechanistic insight into AuNC deligation and provide experimental evidence that protons play an important role in this transformation. Ultimately, this study establishes in operando EIS as a powerful electrochemical characterization tool for monitoring in situ catalyst evolution and deepens our understanding of AuNC deligation behavior.