Microalgal pigments, like chlorophylls, carotenoids, and phycobiliproteins, are valuable bioactive substances with applications in the food, cosmetic, and pharmaceutical industries. They are becoming increasingly popular as a natural alternative to synthetic additives owing to their multifunctional properties, such as antioxidant, anti-inflammatory, and color-enhancing abilities. These pigments are thermolabile and photosensitive; additionally, the complexity of microalgal matrices may pose significant challenges in the efficient extraction and analytical quantification. This chapter reviews the current approaches for developing and validating liquid chromatography-mass spectrometry (LC-MS) protocols for pigment analysis. Key stages of method development include sample preparation, solvent selection, optimized solvent extraction, chromatographic separation, and MS detector selection. The use of reversed-phase and hydrophilic interaction chromatography (HILIC) is highlighted, alongside detector configurations like Q Exactive Orbitrap, triple quadrupole, and quadrupole time-of-flight (Q-TOF), which contribute significant advantages in selectivity, resolution, and throughput. Method validation is crucial to ensure reliability and accuracy in routine practice. In compliance with ICH and FDA guidelines, fundamental method validation criteria for LC-MS include specificity, selectivity, accuracy, linearity, precision, and limits of detection and quantification (LoD/LoQ). This protocol chapter serves as a manual for reliable and reproducible LC-MS workflows for microalgal pigments that benefit both scientific and commercial applications.