Subcellular optogenetics allows specific proteins to be optically activated or inhibited at a restricted subcellular location in intact living cells. It provides unprecedented control of dynamic cell behaviors. Optically modulating the activity of signaling molecules on one side of a cell helps optically control cell polarization and directional cell migration. Combining subcellular optogenetics with live cell imaging of the induced molecular and cellular responses in real time helps decipher the spatially and temporally dynamic molecular mechanisms that control a stereotypical complex cell behavior, cell migration. Here we describe methods for optogenetic control of cell migration by targeting three classes of key signaling switches that mediate directional cellular chemotaxis—G protein coupled receptors (GPCRs), heterotrimeric G proteins, and Rho family monomeric G proteins.