Tropane alkaloids (TAs) are a special class of alkaloids found naturally in a diverse group of flowering plant families. To date, about 200 TAs are known, the most prominent being hyoscyamine, scopolamine, calystegine, and cocaine. These compounds possess pharmacological properties and are used in medicine as anticholinergic agents and stimulants. Because of their medicinal value, tropane alkaloids have been the subject of study for several years now. Over the years, research has been directed at elucidating the biosynthetic pathways leading to the production of pharmacologically active TAs. The present chapter discusses recent developments in the understanding of TA biosynthesis with emphasis on the genes involved in the TA biosynthetic pathways and the role transcriptome profiling played in their identification. In recent years, mining of the transcriptome data of TA-producing plants, such as Atropa belladonna, has led to a near-complete elucidation of the biosynthesis of hyoscyamine and scopolamine. Advances in gene elucidation made through such studies can be potentially used for metabolic engineering in transgenic plant systems or microbial platforms to sustainably meet the global demand of pharmaceutically important TAs.