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Fucoxanthin (FUCO) is extremely instable and occur in nature as different isomers which may differ considerably in their biological effectiveness. Therefore, the aim of this work was to identify the FUCO isomers in oil and powder samples by LCMS and NMR analyses and then to develop a precise, reliable, and rapid HPLCDAD method for quantifying them; moreover, the effect of heating, basic and acid medium and light on their stability was measured. FUCO and isomers were identified as all-trans and 9’-cis fucoxanthin with MS and NMR spectra. Different chromatographic parameters were carefully optimized and the method was specific, linear (1-40 µg mL− 1; r> 0.99), precise (standard deviation values< 0.7%), accurate (mean recovery= 103.25%), low limit of quantification (0.1 µg mL− 1). Moreover, the robustness was investigated using the Box–Behnken experimental design and factors studied simultaneously were: pH of mobile phase, wavelength and column temperature, each at three levels, producing results within the acceptable range. This proposed method was successfully applied the analysis of FUCO, emphasizing thus the advantages of the method: high efficiency, reliable and rapid analysis. Our results suggested that method can be used for the quality control of FUCO and its isomers.