This study aimed to calibrate and validate the MFI-UF method in order to ensure the accuracy of particulate fouling measurements in RO. Firstly, the MFI-UF calibration was examined using two solutions of standard particles (dextran and polystyrene). Two main criteria were investigated: (i) MFI-UF linearity with particle concentrations at both low and high ranges of fouling potential and (ii) the reproducibility of MFI-UF linearity. Dextran solutions showed a strong MFI-UF linearity over the entire range of measured MFI-UF. However, the linearity was not reproducible, and different batches of dextran prepared under the same conditions produced very variable results. For polystyrene solutions, the MFI-UF linearity was verified at the higher range of MFI-UF (>10,000 s/L2), while the MFI-UF at the lower range (<5000 s/L2) appeared to be underestimated. Secondly, MFI-UF linearity was investigated using natural (surface) water under a wide range of testing conditions (at 20–200 L/m2·h using 5–100 kDa membranes). Strong MFI-UF linearity was obtained over the entire range of measured MFI-UF (up to 70,000 s/L2). Thus, the MFI-UF method was validated to measure different levels of particulate fouling in RO. However, future research focusing on MFI-UF calibration is still required through the selection, preparation, and testing of heterogeneous mixtures of standard particles.