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Isochrysis galbana is a marine microalga which highlights by containing a great diversity of antioxidant biomolecules. It shows a high content of lipids that can be used in aquaculture and biofuel, its high polyunsaturated fatty acid profile such as DHA (docosahexaenoic acid) or for having high fucoxanthin content (carotenoid group). For a century, this microalga is very well known as feed for bivalves, larva of fish, crustaceans, and mollusks. Transesterification is the necessary reaction to be able to derivative the fatty acids in methylated fatty acids (AGM) and thus be able to identify and quantify them. It is a key step to optimize and therefore know the improvement of the obtained fatty acid profile of study biomass. Hence, this work presents the significant difference of eight methods for obtaining fatty acids from the direct and indirect transesterification reaction (MTD and MTI, respectively). Moreover, acidic and alkaline catalysts were also used (AC1, AC2 and AL1 and AL2). The results presented better contents of AGMs (methylated fatty acids) respecting dry biomass in the MTD-AL1 method with ~ 6% and a relative abundance of DHA of ~ 12 % in MTI-AL2. In addition, the most abundant fatty acid profile was MTD-AL2 with 57.66 % in polyunsaturated. On the other hand, thanks to the addition of an internal standard in the experiences, it was possible to identify that the MTD-AL1 and MTI-AC1 methods were the most efficient in the transesterification obtained with ~ 93 % and ~ 87 %, respectively. Therefore, the method that is selected for the correct reading of fatty acids present in any biomass is relevant to detect a profile with more unsaturation as has been purchased with the microalga I. galbana. [ABSTRACT FROM AUTHOR]