This chapter explains the steps necessary to perform laser surgery upon single adherent mammalian cells, where individual organelles are extracted from the cells by optical tweezers and the cells are monitored post-surgery to check their viability. Single-cell laser nanosurgery is used in an increasing range of methodologies because it offers great flexibility. Its main advantages are (a) there is not any physical contact with the cells so they remain in a sterile environment, (b) high spatial selectivity so that single organelles can be extracted from specific areas of individual cells, (c) the method can be conducted in the cell’s native media, and (d) in comparison to other techniques that target single cells, such as micromanipulators, laser nanosurgery has a comparatively high throughput.