Cardiovascular and cancer diseases are the first causes of death in the world. Digitalis purpurea L. is a medicinal plant that produces secondary metabolites, like digoxin and digitoxin, which are employed in therapies against heart failure. Moreover, anticancer and antiviral properties of these metabolites have recently been described. The present work details a method to obtain in vitro plants of D. purpurea from leaf segments through direct organogenesis. A reliable and efficient plant induction system was established by optimizing the concentration of naphthaleneacetic acid (NAA) and 6-benzylaminopurine (6-BAP). The highest frequency of shoot regeneration (98.5%) with an average number of shoots per leaf segment of 18.9 was achieved via direct organogenesis from leaf segment on MS medium containing 0.54 μM NAA + 13.2 μM 6-BAP. Additionally, Random Amplified Polymorphic DNA (RAPD) analysis showed 100% monomorphic bands, which indicated genetic stability of the obtained plants. Moreover, leaf powder of de novo regenerated plants fulfills the quality specifications of the British Pharmacopoeia and HPLC analysis revealed the presence of digoxin (22.6 μg gDW −1) and digitoxin (220.7 μg gDW −1) without significant differences in contents between de novo regenerated and mother plants. An efficient in vitro propagation protocol via direct organogenesis and genetic stability assessment of D. purpurea for obtaining leaf powder with quality for the use as raw material have thus been described. The protocol also provides an effective means for several approaches in biotechnology and breeding programs, in order to produce pharmaceutically interesting cardenolides.