This report focuses on measuring the individual electrophoretic mobilities of liposomes with different pH gradients across their membrane using capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). The results from the individual analysis of liposomes show that, using surface electrostatic theories and the electrokinetic theory as the first approximation, ζpotential contributes more significantly to the electrophoretic mobility of liposomes than liposomal size. For liposomes with an outer pH 7.4 (pH o 7.4) and a net negative outer surface charge, the most negative electrophoretic mobilities occur when the inner pH (pHi) is 6.8; at higher or lower pHj, the electrophoretic mobilities are less negative. The theories mentioned above cannot explain these pH-induced electrophoretic mobility shifts. The capacity theory, predicting an induced electrical charge on the surface of liposomes, can only explain the results at pHi > 6.8. In this report, we hypothesize that there is a flip-flop process of phospholipids, which refers to the exchange of phospholipids between the outer and inner layers of the membrane. This flip-flop is caused by the pH gradient and membrane instability and results in the observed electrophoretic mobility changes when pHi is <6.8. Furthermore, it is found that the mobilities of acidic organelles are consistent with the predictions of liposome models we used here.