Bioactive compounds are natural substances found in various plant matrices, known for their antioxidant, antitumor, anti-inflammatory, and antimicrobial properties. The growing demand for natural sources of these compounds has driven research into isolating these substances from plants. This process usually involves several stages: extraction, fractionation, purification, and isolation, using chromatographic techniques such as High-Performance Liquid Chromatography (HPLC), open-column chromatography, and Thin-Layer Chromatography (TLC). These techniques allow for separating and purifying bioactive substances such as flavonoids, alkaloids, and polyphenols. Notable examples of plant-derived bioactives include acetylsalicylic acid from the bark of the willow tree of the Salix genus and morphine from Papaver somniferum. This review explores the methods and steps involved in isolating bioactive compounds from plant matrices, emphasizing the application of chromatography for separating and purifying these substances.