Hydraulic engineers often divide a flow region into subregions to simplify calculations. However, the implementation of flow divisibility remains an open issue and has not yet been implemented as a fully developed mathematical tool for modeling complex channel flows independently of experimental verification. This paper addresses whether a three-dimensional flow is physically divisible, meaning that division lines with zero Reynolds shear stress exist. An intensive laboratory investigation was conducted to carefully measure the time-averaged velocity in a rectangular open channel flow using a laser Doppler anemometry system. Two innovative methods are employed to determine the locations of division lines based on the measured velocity profile. The results clearly reveal that lines with zero total shear stress are discernible, indicating that the flow is physically divisible. Moreover, the experimental data were employed to test previously proposed methods of calculating division lines, and the results show that Yang and Lim's method is the most reasonable predictor.