Modern biology is based largely on a reductionistic "dissection" approach-most cell biologists try to determine how complex biological systems work by removing their individual parts and studying the effects of this removal on the system. A variety of enzymatic and mechanical methods have been developed to dissect large cell assemblies like tissues and organs. Further, individual proteins can be inactivated or removed within a cell by genetic manipulations (e.g., RNAi or gene knockouts). However, there is a growing demand for tools that allow intracellular manipulations at the level of individual organelles. Laser microsurgery is ideally suited for this purpose and the popularity of this approach is on the rise among cell biologists. In this chapter, we review some of the applications for laser microsurgery at the subcellular level and describe practical requirements for laser microsurgery instrumentation demanded in the field. We also outline a relatively inexpensive but versatile laser microsurgery workstation that is being used in our laboratory. Our major thesis is that the limitations of the technology are no longer at the level of the laser, microscope, or software, but instead only in defining creative questions and in visualizing the target to be destroyed.