Current knowledge in the use of hairy root cell cultures is growing. The use of this technology in the production of secondary metabolites is also increasing with the general purpose of generating medicinal alternatives from ethnopharmaceutical origins. Strategies include either genetically modified hairy roots that overexpress genes that codify for key enzymes in complex metabolic pathways or the use of certain elicitors for the natural production of certain compounds in hairy roots. The importance of understanding basic root technology and the complex genetic regulation of root development helps in establishing well-designed strategies in order to obtain higher yields in terms of metabolites or recombinant protein production. Also, bioprocess factors such as the use of bioreactors, elicitors, nutrients, and phytohormones for the induction of compounds of economic importance are also growing. Thus, the aim of the present chapter is to summarize the most important aspects regarding the technology to generate these hairy roots and their main applications in novel fields such as phytoremediation.