Fuel cell performance testing and in operando synchrotron radiography were used to investigate the effect of polytetrafluoroethylene (PTFE) in standalone microporous layers (MPLs) on mass transport and membrane hydration. Two standalone MPLs with 20 wt% and 30 wt% PTFE were fabricated and tested with inlet gas relative humidity (RH) between 50 and 100%. This study demonstrates that the performance of a fuel cell using a standalone MPL with 30 wt% PTFE decreases when the RH of the inlet gases is increased from 50% to 100%, whereas the performance of a fuel cell using a standalone MPL with 20 wt% PTFE remains stable over the same relative humidity range. Furthermore, this study demonstrates that a tradeoff between membrane hydration and mass transport losses must be considered when increasing the PTFE content within the MPL. Higher PTFE content led to greater liquid water accumulation adjacent to the catalyst layer. The greater liquid water accumulation adjacent to the catalyst layer improved membrane hydration and proton conductivity but also led to an increase in mass transport resistance. Standalone MPLs with higher PTFE content did not support high current density operation because the mass transport limitations outweighed the benefits of improved membrane hydration.