Single-cell electroporation (SCE) is a versatile technique for delivering electrically charged macromolecules including DNA, RNA, synthetic oligonucleotides, peptides, dyes, and drugs to individual cells within intact tissues. Here, we describe methods for in vivo-targeted electroporation of single tectal neurons within the albino Xenopus laevis tadpole. Focal electroporation is achieved using a pipette electrode filled with a solution of the delivery molecules and with a tip diameter much smaller than the width of the target cell. The small tip allows for localization of an electric field, which restricts pore formation to only the individual cell in direct contact with the tip. Thus, the small tip permits focal delivery of the charged molecules within the pipette into individual cells. Factors affecting the efficiency of SCE, as well as various applications of this technique, are discussed. Particular focus is directed toward combining SCE with in vivo two-photon microscopy for three-dimensional (3D) imaging of neuron growth and cell-autonomous effects of altered protein function.