Water management is a key factor affecting the efficiency of proton exchange membrane fuel cells (PEMFCs). The currently used monitoring methods of PEMFCs provide limited information about which processes or components that humidity has a significant impact upon. Herein, we propose the use of a novel approach of impedance measurements using a multi-sinusoidal perturbation signal, which enables impedance measurements under dynamic operating conditions. The manuscript presents the effect of the relative humidity (RH) of the reactants on the instantaneous impedance of the middle cell in the PEMFC stack as a function of the current load. Analysis of changes in the values of equivalent circuit elements was carried out to determine which process determines the stack’s performance depending on the load range of the fuel cell during operation. Comprehensive impedance analysis showed that to ensure optimal cell operation, the humidity of the reactants should be adjusted depending on the load level. The results showed that at low-current loads, the humidity of gases should be at least 50%, while at high-current loads, the cell should operate optimally at a gas humidity of 30% or lower. The presented methodology provides an important tool for optimizing and monitoring the operation of fuel cells.