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Microalgae with high growth rates represent a potential source of carotenoids including fucoxanthin. For commercial fucoxanthin production, choosing the cultivation system is a critical parameter where the final biomass must contain a high content of the target product. To realize this, the reaction system must allow the use of stress conditions that induce the synthesis of these compounds. In this study, oxidative stress was applied to the diatom Amphora capitellata by the formation of reactive oxygen species in the culture medium through chemical reactions which generate. The effect of this oxidative stress on the amount of fucoxanthin and specific growth rate of A. capitellata was investigated. In the presence of H2O2/NaOCl, fucoxanthin content was 41.83 ± 0.92 mg g−1 as determined by HPLC–DAD a 2.20-fold increase compared with the control group. For the purification of fucoxanthin, preparative HPLC using a semi-prep C30 carotenoid column (10 × 250 mm, 5 µm) was used for the first time with an isocratic elution of 75:25 (methanol:acetonitrile) mobile phase at a flow rate of 4 mL min−1. Liquid chromatography tandem mass spectrometry (LC–MS/MS) was employed for the confirmation of fucoxanthin. The developed method has potential to obtain highly pure fucoxanthin for food and pharmaceutical applications using A. capitellata as a commercial source of fucoxanthin, particularly after its enhancement via oxidative stress. [ABSTRACT FROM AUTHOR]