The pigments derived from microalgae, specifically phycobiliproteins, carotenoids, and chlorophylls, have unique physiological effects and health effects in humans. Due to their vibrant colors, antibacterial, antioxidant, and UV-protective properties, these pigments are widely used in the food, pharmaceutical, and cosmetic industries. Microalgal pigment research is becoming increasingly popular due to its high pigment productivity, current industrial-scale manufacturing, more sustainable production methods, and absence of seasonal dependence. The most widely used separation method for identifying, isolating, and measuring bioactive substances, drugs, pigments, etc., is high-performance liquid chromatography (HPLC). The main aim of the chapter is to describe the protocol for HPLC analysis of microalgal pigments. Optimizing the gradient, run time, injection volume, column temperature, and composition of the mobile phase is also essential. A number of chromatographic parameters, including sample preparation, mobile phase selection, column selection, and detector selection, are standardized in order to optimize the procedure. Here, in this chapter, we have discussed the methods of sample preparation, extraction of microalgal pigments through Heat-assisted extraction (HAE), Microwave-assisted extraction (MAE), and Ultrasound-assisted extraction (UAE), and the HPLC analysis method of the isolated pigments and data analysis.