Effective water management is required to enable polymer electrolyte membrane fuel cells (PEMFCs) to operate efficiently, particularly at the high current densities needed to make them relevant for transportation and stationary applications. This study was conducted to evaluate the effects of selected operating conditions on the performance of PEMFCs, as well as their influence on the distribution of liquid water within specific fuel cell components. The investigation included assessments of the impact of operating temperature, oxidant concentration gas delivery rates, relative humidity of influent gases, and current density on fuel cell (FC) performance and water distribution. Miniaturized PEMFCs were constructed, and their performance was assessed using three main electrochemical diagnostic tools, including polarization curve measurements, cyclic voltammetry, and electrochemical impedance spectroscopy. Liquid water distribution and volume were determined in-operando using X-ray computed tomography-based procedures and associated image processing methods. The results of this study indicated that the performance of PEMFCs can be substantially affected by varying operational conditions, including test station setup, influent gas temperature and flow rates, oxidant concentration (i.e., air vs O₂ at varying concentrations), with the best performance observed at 70°C, high influent gas flow rates, and high oxygen concentrations. Water distribution within the membrane electrode assembly was influenced by current density, operating temperature, influent gas flow rates, and oxidant type. Information on the distribution of liquid water was useful for explaining differences in FC performance under various operating conditions (e.g., 40 °C compared to 70°C). The results of this work provide useful information for optimizing the performance of PEMFCs and enhancing their uses in target applications.