Dendritic spines are major sites to receive synapses in the mammalian brain. Spines with abnormal morphologies are found in different brain diseases, suggesting that malformation of dendritic spines could be causally linked to those diseases. Rho GTPase-signaling pathways are implicated in the regulation of spine morphology and also in some forms of mental retardation. Therefore, understanding the dynamic regulation of spine morphology by Rho GTPases may provide insights into the etiology and therapeutic strategy of brain diseases. This chapter describes methods used to examine the molecular mechanisms regulating the morphological features of dendritic spines, including slice cultures, biolistic transfections, and live imaging techniques, and summarizes our findings made using these methods.