Due to the importance of chlorophyll in phytoplankton, there are a number of analytical protocols. In this regard, the current chapter aims to be a practical guide for scientists interested in the description of the methodologies applicable to studying chlorophylls in phytoplankton. The complex pigment profile and the intricacies of pigment extraction from phytoplankton are the two main challenges encountered during chlorophyll analysis. The cell walls of certain species hinder the solvent capacity. For instance, Cyanobacteria and certain chlorophytes are termed recalcitrant. The current chapter will review (a) the different techniques that have been applied to guarantee the exhaustive chlorophyll extraction from microalgae and cyanobacteria, (b) the methods employed for chlorophyll analysis: which are often analyzed by liquid chromatography coupled with a diode array detector (DAD). Diverse chromatographic methods have been optimised considering different structures of the chlorophyll derivatives present in phytoplankton. A deeper analysis has been achieved through the mass spectrophotometric (MS) studies of chlorophyll compounds of main microalgae and cyanobacteria, including the analysis of the fragmentation pathways to increase the consistency of the identification. However, the disposal of appropriate standards is essential in chlorophyll investigation irrespective of the employed method of analysis. Only a few chlorophyll standards are commercially available, and consequently, protocols for yielding the others have been specifically developed. Finally, the chapter will summarize the main chromatographic, spectroscopic properties, and MS characteristics of all chlorophyll present in phytoplankton.