The resistance-in-series (RIS) model should be used cautiously, particularly in the experimental determination of the cake layer resistance (R-c) which is determined by calculation of a series of flux data that are obtained empirically before and after removing the cake layer on the membrane surface. However, the calculated R-c values are very dependent upon the cleaning methods used for removing the cake layer. Therefore, this study investigated how the various cleaning methods influence the determination of R-c. Four different cleaning methods were used: water rinsing in a shaker, manual water rinsing, ultrasonication, and sponge scrubbing. For the hydrophilic membrane, sponge scrubbing removed the cake layer completely, whereas the other methods showed removal efficiencies ranging from 79% to 99%. For the hydrophobic membrane, none of the options achieved complete cake layer removal. In addition, sponge scrubbing was not the best option for cake removal, indicating that even a method with the potential to completely remove the cake layer on a specific membrane is not universal for every kind of membrane. Consequently, a standardized method for cake layer removal to determine cake resistance (R-c) is needed for correct interpretation of the fouling phenomena with the RIS model.