Fluorescence recovery after photobleaching (FRAP) can be employed to investigate membrane lipid mixing of vacuoles in live budding yeast cells and distinguish the fused, hemi-fused or non-fused states of these organelles under physiological conditions. Here, we describe a protocol for labeling the outer and inner leaflets of vacuoles in live cells that allow to detect hemifusion intermediates and, thus, identify components necessary for fusion pore opening.