Phytochemicals derived from plants play a crucial role in promoting human health. Plants are rich sources of essential nutrients, including proteins, fats, vitamins, minerals, carbohydrates, and various bioactive compounds with significant nutritional and therapeutic value. Despite their importance, many plant-derived compounds remain unexplored by the scientific community. Therefore, effective methods for the screening and characterization of phytochemicals—such as phenolics, alkaloids, and flavonoids—are essential to unlock their full potential. Chromatographic techniques, particularly High-Performance Liquid Chromatography (HPLC), have proven highly effective for the qualitative and quantitative analysis of phytochemicals. A more advanced approach, HPLC coupled with a Diode Array Detector (HPLC-DAD), enhances this process by combining the efficient separation capabilities of HPLC with the ability to detect and identify compounds based on their UV-Visible absorption spectra. This method is especially effective for analyzing complex mixtures, including plant alkaloids, pharmaceuticals, and other bioactive compounds. This chapter focuses on the application of HPLC-DAD in the screening and characterization of plant-derived alkaloids.