Background: In a precedent paper, we validated part IV of the Unified Parkinson’s Disease Rating Scale (UPDRS) for detecting motor fluctuations in Parkinson’s Disease (PD) patients using a 12-h Waking-Day Motor Assessment (WDMA) as gold standard, showing a high sensitivity (> 80%) and a lower specificity (< 45%). The aim of this study was to validate the Movement Disorder Society-UPDRS (MDS-UPDRS) part IV, especially items 4.3 and 4.5, using the same methodology. Methods: PD patients attending the Movement Disorders Clinic at the University Hospital in Catania were consecutively enrolled in the study. A diurnal WDMA was performed to detect motor fluctuations. At each time interval, the motor impairment was evaluated using the motor section of the MDS-UPDRS. Presence or absence of motor fluctuations and the type of motor fluctuation were assessed by four blinded expert raters in movement disorders, by evaluating the graphical representations of the WDMA. We evaluated sensitivity and specificity together with 95% Confidence Interval (CI) of items 4.3 and 4.5, using WDMA as gold standard. Results: We estimated for item 4.3 of the MDS-UPDRS a sensitivity of 74.3% (95% CI 56.7–87.5) and a specificity of 70.6% (95% CI 44–89.7), while for item 4.5, a sensitivity of 67.9% (95% CI 47.6–84.1) and a specificity of 66.7% (95% CI 44.7–84.4). Conclusions: The present showed a higher specificity level for MDS-UPDRS with respect to the UPDRS, while a slightly lower sensitivity mainly for predictable OFF.