The microalgal biomass present ascendant commercial importance for food industry, pharmaceutical, energy and aquaculture as a natural source of high value-added products, pigments, enzymes, lipids, proteins, sterols and vitamins. However, no data are reported about precursor metabolites and biosynthesis intermediates of microalgae compounds. The purpose of this study was to develop of a simultaneous extraction method for polar (PC - amino acids and organic acids) and non-polar compounds (NPC - fatty acids). Additionally, determination method for these compounds was validated and used for characterization of two Chlorophyceae, Chlorella vulgaris and Scenedesmus obliquus and two Cyanobacteria, Aphanothece microscopica Nagëli and Phormidium autumnale. The compounds were extracted by organic solvent mixture (methanol-chloroform) with 80% less organic solvents and sample and different forms of agitation and/or cell disruption. The NPCs were derivatized by acid catalysis. Derivatization procedure of PC fraction was made using N-methyl, N-tert-Butyldimethylsilyltrifluoroacetamide (MTBSTFA) in alkaline medium for biomass microalgae samples. The method validation of PC considered selectivity, linearity, limit of detection (LOD), limit of quantitation (LOQ), precision and accuracy. All methods homogenization and/or cell disruption showed ability to extract polar and non-polar compounds from Chlorophyceae and Cyanobacteria. Derivatization of polar compounds presented satisfactory parameters of validation. Fourteen fatty acids, fifteen amino acids and three organic acids were found among species. Succinic, malic and citric acids, important intermediates of tricarboxylic acid cycle (TCA), so it was possible to establish a relationship between this cycle and the Calvin cycle and microalgal interest products. The high concentrations of glutamic acid for all species evaluated may also be related to an important derivative, 5-aminolevulinic acid, precursor of porphyrin tetrapyrroles, such as vitamin B12 and chlorophyll. The Chlorophyceae presented a predominantly unsaturated fatty acid profile while the Cyanobacteria, saturated. Thus, extraction and derivatization method was efficient for metabolic characterization of Chlorophyceae and Cyanobacteria.