One of the most important index in oceanography, phytoplankton biomass, is based on determination of chlorophyll a, an algae pigment. Unlike higher plants, algae show in their composition a large number of pigments broadly called accessory or photosynthetic pigments. These pigments include chlorophylls, carotenoids and phycobiliproteins. Measuring the relative or absolute concentration of algae pigments is a powerful tool to study algae phylogeny, taxonomy, physiology, ecology and others. Chlorophyll a concentration is determined in natural samples either by spectroscopy or fluorometry. In the other hand, the determination of other photosynthetic pigments, such as carotenoids, is difficult due to their close absorption characteristic and the absence of fluorescence. In the last few years, with new materials and technologies development, chromatographic methods, specially high performance liquid chromatography (HPLC), have been successfully applied to quantify chlorophylls and carotenoids in natural waters. These new meth¬odologies opened a new perspective on measuring algae pigments, either by improving the determination of chlorophyll a precision or by making possible to measure up to 50 pigments, including some degradation products. In this paper, a procedure to qualitatively calibrate an HPLC system is presented. Nineteen pigments, from six species, representing different algae groups (dinoflagellates, green algae, cryptomonads, prymnesiophytes, diatoms and cyanophytes) were separated and identified by their retention times and spectral characteristics. The method application is exemplified in 3 natural samples.