Phytosterols, plant-derived sterols, have attracted significant attention for their potential health benefits. They exert antioxidant, anti-inflammatory, and anticancer activities for which they have been considered a promising candidate to be used as functional food. A multitude of phytosterols like β-sitosterol, campesterol, and stigmasterol have been investigated for their therapeutic potential. This chapter aims to describe the methodologies used for isolation and characterization of phytosterols. The isolation process typically involves the extraction of plant materials using organic solvents, followed by purification using chromatographic methods. Advanced chromatographic methods, such as high-performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS), have been employed to ensure the purity and structural integrity of phyosterols. Additionally, spectroscopic techniques, including nuclear magnetic resonance (NMR) and infrared (IR) spectroscopy, have also been used for detailed structural characterization. This chapter consolidates methodologies for isolation and characterization of phytosterols in order to fully harness their therapeutic potential.