Fruits of chili peppers (Capsicum spp.) specifically synthesize and accumulate a group of analogs known as capsaicinoids in the placenta tissues. These secondary metabolites are responsible for the hot taste of chili pepper fruits. Capsaicinoids are of economic importance because of their use in the food, cosmetic, military, and pharmaceutical industry. Several efforts have been focused to investigate the biosynthetic capacity of in vitro chili pepper cells and tissue cultures in order to determine the production feasibility of these compounds at the industrial level under controlled conditions. A description of techniques for the establishment of in vitro cultures of chili pepper, the addition of precursors and intermediates to the culture medium, and the selection of cell lines as a means to increase the production of capsaicinoids as well as the extraction, separation, and quantification of capsaicinoids from chili pepper cell cultures is reported in this chapter.