Alkaloids, a diverse group of naturally occurring nitrogen-containing compounds, are renowned for their wide-ranging health-promoting properties, including anticancer, anti-inflammatory, antimicrobial, and neuroprotective effects. Owing to their therapeutic potential, the efficient isolation and accurate characterization of alkaloids from plant and microbial sources are of critical importance in drug discovery and nutraceutical development. This chapter presents a comprehensive overview of classical and advanced methodologies for alkaloid isolation and purification by column chromatography and high-speed counter-current chromatography. Furthermore, it highlights the characterization strategies employed through various chromatographic techniques like TLC, HPLC, UPLC, and GC-MS. Emphasis is placed on the analytical precision, instrumentation setups, and procedural optimizations required for the structural elucidation and quantification of bioactive alkaloids. By bridging phytochemical isolation techniques with modern analytical chemistry, this chapter aims to facilitate the application of health-benefiting alkaloids in the biomedical and pharmaceutical sectors.