The centrosome is positioned between the nucleus and the leading edge of many types of migrating cells. Cdc42 regulates this centrosome reorientation through its effectors Par6 and MRCK. Using time-lapse microscopy of live cells, the mechanisms and kinetics of centrosome reorientation can be studied. In this chapter, we describe a modification in the standard wound healing assay that allows the study of signaling pathways involved in centrosome reorientation and other polarization events that occur before cell migration. We also describe a method for visualization of centrosome reorientation by time-lapse microscopy using NIH 3T3 fibroblasts stably transfected with GFP-tubulin.