Context:Quercetin (QR) is a natural strong antioxidant of the plant origins. It is used for treating conditions of heart and blood vessels including “hardening of arteries” (atherosclerosis), high cholesterol, heart disease, circulation problems, preventing cancer, for treating chronic infections of the prostate, etc.,Aims:The aim of this work was to development and study of an acid-free technique for the preparation of QR from rutin (RT) that requires no use of acids and toxic organic solvents.Materials and Methods:For the first time, the subcritical water that serves as a reactant and a solvent were used to obtain QR in good yields starting from RT. High-performance liquid chromatography combined with mass spectrometry was used to determine the quantitative and qualitative compositions of the obtained products.Conclusions:For the first time, a new acid-free technique was used for the synthesis of natural antioxidant QR from an RT by subcritical water. That way requires no use of acids and/or toxic organic solvents. It has been shown that variation of only one parameter of the process (temperature of subcritical water) allows alteration to the composition of the hydrolysis products. The new method developed for the production of QR in subcritical water is environmentally friendly and faster than conventional hydrolysis methods that use acidic or enzymatic hydrolysis. The proposed technique has a potential for the future development of inexpensive and environmentally friendly technologies for the production of new pharmaceutical plant-based substances.