Known for their diverse and potent physiological activities, natural products continue to be essential for the discovery and development of new drugs. This chapter explores the pivotal role of preparative thin-layer chromatography (Prep-TLC) in the isolation of natural products. This chapter begins with an understanding of the historical significance and structural complexity of natural products, and discusses the problems caused by complex mixtures present in extracts, as well as the multifunctionality, cost-effectiveness, and compatibility with different sample types of Prep-TLC to address these challenges. We then explain the problems of separating these compounds and introduce Prep-TLC as a solution. The focus shifts to the fundamental principles and methodologies of Prep-TLC, highlighting its role in achieving the efficient separation and isolation of natural products. To illustrate the successful application of Prep-TLC to isolate bioactive compounds from various natural sources, real-world case and case studies are presented. In this chapter, we aim to provide practical assistance to researchers and practitioners by explaining the principles and methodologies of Prep-TLC that facilitate the discovery of novel physiologically active compounds.