Many studies in modern biology often rely on the introduction of a foreign molecule (i.e., transfection), be it DNA plasmids, siRNA molecules, protein biosensors, labeled tracers, and so on, into cells in order to answer the important questions of today's science. Many different methods have been developed over time to facilitate cellular transfection, but most of these methods were developed to work with a specific type of molecule (usually DNA plasmids) and none work well enough with difficult, sensitive, or primary cells to meet the needs of current life science researchers. A novel procedure that uses laser light to gently permeabilize large number of cells in a very short time has been developed and is described in detail in this chapter. This method allows difficult cells to be efficiently transfected in a high-throughput manner, with a wide variety of molecules, with extremely low toxicity.