Trioxidane can be obtained in small, but detectable, amounts in reactions of ozone and hydrogen peroxide, or by the electrolysis of water. Larger quantities have been prepared by the reaction of ozone with organic reducing agents at low temperatures in a variety of organic solvents, such as the anthraquinone process. It is also formed during the decomposition of organic hydrotrioxides (ROOOH). Alternatively, trioxidane can be prepared by reduction of ozone with 1,2-diphenylhydrazine at low temperature.  Using a resin-bound version of the latter, relatively pure trioxidane can be isolated as a solution in organic solvent. Preparation of high purity solutions is possible using the methyltrioxorhenium(VII) catalyst. In acetone-d at −20 °C, the characteristic H NMR signal of trioxidane could be observed at a chemical shift of 13.1 ppm. Solutions of hydrogen trioxide in diethyl ether can be safely stored at −20 °C for as long as a week.
The reaction of ozone with hydrogen peroxide is known as the "peroxone process". This mixture has been used for some time for treating groundwater contaminated with organic compounds. The reaction produces HO and HO.