Tylophora, a genus of the family Asclepiadaceae, is common in the Indian subcontinent and other parts of Asia, such as Sri Lanka, Malaysia, Thailand, Australia, and Africa. Since ancient times, the plants of this genus have been utilized as medicines to treat diseases such as arthritis, gout, jaundice, constipation, flatulence, hemorrhoids, whooping cough, asthma, congestion, and inflammatory skin. The most critical phytochemicals of Tylophora that are accountable for their medicinal application are phenanthroindolizidine alkaloids, which are present primarily in aerial parts of plants. The alkaloids are isolated using different extraction and chromatographic techniques, including HPLC. Alkaloids have been investigated for potential therapeutic applications such as cytotoxic, anticancer, antimicrobial, and antiproliferative. The book chapter overviews Tylophora alkaloids and their structure, isolation, and biological applications.