A common procedure includes the dropwise addition of concentrated sulfuric acid to a cooled mixture of an aqueous sodium nitrite solution and an alcohol.

Amyl nitrite may be used during cardiovascular stress testing in patients with suspected hypertrophic cardiomyopathy to cause vasodilation and thereby reduce afterload and provoke obstruction of blood flow towards the aorta from the ventricle by increasing the pressure gradient, thereby causing left ventricular outflow obstruction.

The ampule is put in a gauze pad and then inhaled by the patient during an angina attack and repeated every fifteen minutes.

However, oral dosing of amyl nitrite is ineffective due to poor absorption and extensive hepatic metabolism.

Although there are case reports of life-threatening toxicity involving unusually large amounts,[21] typical inhaled doses of amyl nitrite are considered relatively safe.

Severe poisoning cases will progress to methemoglobinemia, characterized by a blue-brown discoloration under the skin which could be mistaken for cyanosis.

[21] Therefore, the measurement of actual ratios and levels of methemoglobin and hemoglobin must accompany any blood gas partial pressure sample in these cases.

The Columbo episode titled "Troubled Waters" (1974–1975) features amyl nitrite inhaled by the antagonist Hayden Danziger – played by Robert Vaughn – to help him feign a heart attack for his alibi.

[27] The Hunter S. Thompson book Fear and Loathing in Las Vegas sees amyl nitrite as one of the many drugs Raoul Duke packs for the trip to Las Vegas, taking about two dozen ampules of it with him and usually justifying its usage by him and Dr. Gonzo to other people around them by claiming it is for angina.