Considering the high interest and commercial value of pigments from microalgae, this study aimed to stablish the best lutein and β-carotene extraction method for Desmodesmus sp., a microalgae with high biomass production. The evaluation was done considering the best solvent or solvent mixture, number of extractions, pre-treatment of dry biomass with acetone, saponification in methanol and ethanol and stability evaluation of the obtained extract. Among achieved results, the best solvent extractor was a hexane:ethanol mixture in a 1:1 (v/v) proportion. In order to obtain maximum yield, the pattern stablished includes six extractions performed for 40 minutes. Stability tests presented that xanthophylls and carotenes pigments were more stable in methanol and that xanthophylls and carotenes pigments were less negatively affected than chlorophylls in the methanolic extract. Chromatographic identification and quantification of pigments from Desmodesmus sp. microalgae revealed that its major constituents are trans-zeaxanthin, chlorophyll b, β-carotene and trans-lutein.