Purpose: Perineal electromyography (EMG) is a crucial part of urodynamic studies. Many researchers focused on the standardization of techniques in urodynamics, but no study has yet evaluated the differences with various reference electrode placements. The aim of this study was to determine the optimal placement of the reference electrode for recording pelvic floor activity in urodynamic studies. Methods: Children over 6 years of age without anatomic or neurological abnormalities were invited to participate in the study. Four reference electrodes were placed on the right kneecap, the inner surface of the right inner thigh, the right anterior iliac spine (AIS), and the skin over the left gluteal muscles for simultaneous recording. The EMG signal formed by pelvic contraction during forceful straining was recorded both in the supine and sitting positions. The root mean square (RMS) value of each muscle contraction signal was calculated. Results: Twenty-one participants (10 boys and 11 girls) were included. The mean age was 10.19±3.20 years. The highest RMS values were obtained with the reference electrode on the thigh in the supine position and the AIS in the sitting position. Significant differences were found between the mean RMS values of the knee and other locations of reference electrodes in the supine position, as well as between mean RMS values in all regions except the thigh and gluteus in the sitting position. The minimum mean RMS values in both positions were obtained with the reference electrode on the knee. Conclusions During urodynamic studies, reference electrodes shall be placed on AIS in the sitting and on the inner thigh in the supine position. The knee is not a suitable option for reference electrode placement. This information may help improve EMG recordings in the evaluation of pelvic floor muscles.