Algae are an interesting source of bioactive compounds, and their polymeric carbohydrate fraction can be broken down to their monomers. In addition, Rugulopteryx okamurae is an invasive alga that it is difficult to remove from coasts. The objective of this work is to validate an analytical method and develop an environmentally friendly technique to quantify algal monosaccharides. Gas chromatography coupled with tandem mass spectrometry (QTOF) has been used to identify monosaccharides, using derivatization with oximes and sylilation. The internal standard was inositol. Pretreatment consisted of milling the algae and make a pretreatment using ethanol to eliminate pigments and phenolic compounds. The solvent used in this green extraction was water, and the extraction technique was ultrasound-assisted extraction (UAE). The first optimization concerned the mass–solvent ratio, testing between 1:15 and 1:30; 1:15 was selected as optimum. UAE has been optimized by experimental design, evaluating power and time in ranges of 20-70% power and 30-120 min by a factorial design. The maximum was established at 70% power and 30 minutes. The method is selective, sensitive and accurate. Rugulopteryx okamurae has mainly manitol, and other monosaccharides are galactose and glucose. Ulva ohnoi possess xylose, ramnose, galactose and glucose. A green and GRAS method was developed to extract monosaccharide from invasive algae.